scholarly journals Shigella flexneri Disruption of Cellular Tension Promotes Intercellular Spread

Cell Reports ◽  
2020 ◽  
Vol 33 (8) ◽  
pp. 108409
Author(s):  
Jeffrey K. Duncan-Lowey ◽  
Alexandra L. Wiscovitch ◽  
Thomas E. Wood ◽  
Marcia B. Goldberg ◽  
Brian C. Russo
Keyword(s):  
Shigella Flexneri ◽  
Intercellular Spread

scholarly journals Requirement for Formin-Induced Actin Polymerization during Spread of Shigella flexneri

2009 ◽  
Vol 78 (1) ◽  
pp. 193-203 ◽  
Author(s):  
Jason E. Heindl ◽  
Indrani Saran ◽  
Chae-ryun Yi ◽  
Cammie F. Lesser ◽  
Marcia B. Goldberg
Keyword(s):  
Plasma Membrane ◽  
Virus Infection ◽  
Vaccinia Virus ◽  
Cell Body ◽  
Actin Polymerization ◽  
Shigella Flexneri ◽  
Microbial Pathogens ◽  
Bacterial Surface ◽  
Virulence Protein ◽  
Intercellular Spread

ABSTRACT Actin polymerization in the cytosol and at the plasma membrane is locally regulated by actin nucleators. Several microbial pathogens exploit cellular actin polymerization to spread through tissue. The movement of the enteric pathogen Shigella flexneri, both within the cell body and from cell to cell, depends on actin polymerization. During intercellular spread, actin polymerization at the bacterial surface generates protrusions of the plasma membrane, which are engulfed by adjacent cells. In the cell body, polymerization of actin by Shigella spp. is dependent on N-WASP activation of the Arp2/Arp3 complex. Here we demonstrate that, in contrast, efficient protrusion formation and intercellular spread depend on actin polymerization that involves activation of the Diaphanous formin Dia. While the Shigella virulence protein IpgB2 can bind and activate Dia1 (N. M. Alto et al., Cell 124:133-145, 2006), its absence does not result in a detectable defect in Dia-dependent protrusion formation or spread. The dependence on the activation of Dia during S. flexneri infection contrasts with the inhibition of this pathway observed during vaccinia virus infection.

scholarly journals Selection for phage resistance reduces virulence of Shigella flexneri .

2021 ◽  
Author(s):  
Kaitlyn E. Kortright ◽  
Rachel E. Done ◽  
Benjamin K. Chan ◽  
Valeria Souza ◽  
Paul E. Turner
Keyword(s):  
Genome Sequencing ◽  
Phage Therapy ◽  
Shigella Flexneri ◽  
Cell Membrane Permeability ◽  
Phage Resistance ◽  
Trade Off ◽  
Trade Offs ◽  
Resistant Mutants ◽  
Selection For ◽  
Intercellular Spread

There is increasing interest in phage therapy as an alternative to antibiotics for treating bacterial infections, especially using phages that select for evolutionary trade-offs between increased phage resistance and decreased fitness traits such as virulence in target bacteria. A vast repertoire of virulence factors allows the opportunistic bacterial pathogen, Shigella flexneri , to invade human gut epithelial cells, replicate intracellularly, and evade host immunity through intercellular spread. It is previously shown that OmpA is necessary for intercellular spread of S. flexneri . We hypothesized that a phage which uses OmpA as a receptor to infect S. flexneri , should select for phage-resistant mutants with attenuated intercellular spread. Here we show that phage A1-1, requires OmpA as a receptor and selects for reduced virulence in S. flexneri . We characterized five phage-resistant mutants by measuring phenotypic changes in various traits: cell-membrane permeability, total lipopolysaccharide (LPS), sensitivity to antibiotics, and susceptibility to other phages. Results separated the mutants into two groups: R1 and R2 phenotypically resembled ompA knockouts, whereas R3, R4 and R5 were similar to LPS-deficient strains. Whole genome sequencing confirmed that R1 and R2 had mutations in ompA , while R3, R4 and R5 showed mutations in LPS inner-core biosynthesis genes gmhA and gmhC . Bacterial plaque assays confirmed that all phage-resistant mutants were incapable of intercellular spread. We concluded that selection for S. flexneri resistance to phage A1-1 generally reduced virulence (i.e. intercellular spread), but this trade-off could be mediated either by mutations in ompA or in LPS-core genes that likely altered OmpA conformation. Author Summary Shigella flexneri is a facultative intracellular pathogen of humans, and a leading cause of bacillary dysentery. With few effective treatments and rising antibiotic resistance in these bacteria, there is increasing interest in alternatives to classical infection management of S. flexneri infections. Phage therapy poses an attractive alternative, particularly if a therapeutic phage can be found that results in an evolutionary trade-off between phage resistance and bacterial virulence. Here, we isolate a novel lytic phage from water collected in Cuatro Cienegas, Mexico that uses the OmpA porin of S. flexneri as a receptor. We use phenotypic assays and genome sequencing to show that phage A1-1 selects for phage-resistant mutants that can be grouped into two categories: OmpA-deficient mutants and LPS-deficient mutants. Despite these underlying mechanistic differences, we confirmed that naturally-occurring phage A1-1 selected for evolved phage resistance that coincided with impaired intercellular spread of S. flexneri in a eukaryotic infection model.

scholarly journals Vaccination against Shigellosis with Attenuated Shigella flexneri 2a Strain SC602

1999 ◽  
Vol 67 (7) ◽  
pp. 3437-3443 ◽  
Author(s):  
Trinka S. Coster ◽  
Charles W. Hoge ◽  
Lillian L. VanDeVerg ◽  
Antoinette B. Hartman ◽  
Edwin V. Oaks ◽  
...  
Keyword(s):  
Shigella Flexneri ◽  
Virulence Gene ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dose Selection ◽  
Chromosomal Locus ◽  
Antibody Secreting Cell ◽  
Severe Diarrhea ◽  
Shigella Flexneri 2A ◽  
Intercellular Spread ◽  
Mild Diarrhea

ABSTRACT The Shigella flexneri 2a SC602 vaccine candidate carries deletions of the plasmid-borne virulence gene icsA(mediating intra- and intercellular spread) and the chromosomal locusiuc (encoding aerobactin) (S. Barzu, A. Fontaine, P. J. Sansonetti, and A. Phalipon, Infect. Immun. 64:1190–1196, 1996). Dose selection studies showed that SC602 causes shigellosis in a majority of volunteers when 3 × 108 or 2 × 106 CFU are ingested. In contrast, a dose of 104 CFU was associated with transient fever or mild diarrhea in 2 of 15 volunteers. All volunteers receiving single doses of ≥104 CFU excreted S. flexneri 2a, and this colonization induced significant antibody-secreting cell and enzyme-linked immunosorbent assay responses against S. flexneri 2a lipopolysaccharide in two-thirds of the vaccinees. Seven volunteers who had been vaccinated 8 weeks earlier with a single dose of 104 CFU and 7 control subjects were challenged with 2 × 103 CFU of virulent S. flexneri 2a organisms. Six of the control volunteers developed shigellosis with fever and severe diarrhea or dysentery, while none of the vaccinees had fever, dysentery, or severe symptoms (P = 0.005). Three vaccinees experienced mild diarrhea, and these subjects had lower antibody titers than did the fully protected volunteers. Although the apparent window of safety is narrow, SC602 is the first example of an attenuated S. flexneri 2a candidate vaccine that provides protection against shigellosis in a stringent, human challenge model.

scholarly journals dksA Is Required for Intercellular Spread of Shigella flexneri via an RpoS-Independent Mechanism

2001 ◽  
Vol 69 (9) ◽  
pp. 5742-5751 ◽  
Author(s):  
Scott A. Mogull ◽  
Laura J. Runyen-Janecky ◽  
Mei Hong ◽  
Shelley M. Payne
Keyword(s):  
Epithelial Cells ◽  
Cultured Cells ◽  
Shigella Flexneri ◽  
Cell Cytoplasm ◽  
Independent Mechanism ◽  
Virulence Protein ◽  
Intercellular Spread ◽  
Mutant Cells ◽  
And Oxidative Stress ◽  
Intracellular Analysis

ABSTRACT Pathogenesis of Shigella flexneri is dependent on the ability of the bacterium to invade and spread within epithelial cells. In this study, we identified dksA as a gene necessary for intercellular spread in, but not invasion of, cultured cells. The S. flexneri dksA mutant exhibited sensitivity to acid and oxidative stress, in part due to an effect of DksA on production of RpoS. However, an S. flexneri rpoS mutant formed plaques on tissue culture monolayers, thus excluding DksA regulation of RpoS as the mechanism responsible for the inability of the dksA mutant to spread intercellularly. Intracellular analysis of the dksA mutant indicates that it survived and divided within the Henle cell cytoplasm, but thedksA mutant cells were elongated, and some exhibited filamentation in the intracellular environment. Some of the S. flexneri dksA mutant cells showed aberrant localization of virulence protein IcsA, which may inhibit spread between epithelial cells.

scholarly journals IpaD of Shigella flexneri Is Independently Required for Regulation of Ipa Protein Secretion and Efficient Insertion of IpaB and IpaC into Host Membranes

2005 ◽  
Vol 73 (3) ◽  
pp. 1432-1440 ◽  
Author(s):  
Wendy L. Picking ◽  
Hiroaki Nishioka ◽  
Patricia D. Hearn ◽  
M. Aaron Baxter ◽  
Amanda T. Harrington ◽  
...  
Keyword(s):  
Protein Secretion ◽  
Shigella Flexneri ◽  
Erythrocyte Membranes ◽  
Bacterial Invasion ◽  
Cellular Invasion ◽  
Host Cytoplasm ◽  
Host Membrane ◽  
Direct Outcome ◽  
Intercellular Spread

ABSTRACT Shigella flexneri causes human dysentery after invading the cells of the colonic epithelium. The best-studied effectors of Shigella entry into colonocytes are the invasion plasmid antigens IpaC and IpaB. These proteins are exported via a type III secretion system (TTSS) to form a pore in the host membrane that may allow the translocation of other effectors into the host cytoplasm. TTSS-mediated secretion of IpaD is also required for translocation pore formation, bacterial invasion, and virulence, but the mechanistic role of this protein is unclear. IpaD is also known to be involved in controlling Ipa protein secretion, but here it is shown that this activity can be separated from its requirement for cellular invasion. Amino acids 40 to 120 of IpaD are not essential for IpaD-dependent invasion; however, deletions in this region still lead to constitutive IpaB/IpaC secretion. Meanwhile, a central deletion causes only a partial loss of control of Ipa secretion but completely eliminates IpaD's invasion function, indicating that IpaD's role in invasion is not a direct outcome of its ability to control Ipa secretion. As shigellae expressing ipaD N-terminal deletion mutations have reduced contact-mediated hemolysis activity and are less efficient at introducing IpaB and IpaC into erythrocyte membranes, it is possible that IpaD is responsible for insertion of IpaB/IpaC pores into target cell membranes. While efficient insertion of IpaB/IpaC pores is needed for optimal invasion efficiency, it may be especially important for Ipa-dependent membrane disruption and thus for efficient vacuolar escape and intercellular spread.

scholarly journals The Vps/VacJ ABC Transporter Is Required for Intercellular Spread of Shigella flexneri

2013 ◽  
Vol 82 (2) ◽  
pp. 660-669 ◽  
Author(s):  
Chandra D. Carpenter ◽  
Benjamin J. Cooley ◽  
Brittany D. Needham ◽  
Carolyn R. Fisher ◽  
M. Stephen Trent ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Outer Membrane ◽  
Abc Transporter ◽  
Shigella Flexneri ◽  
Dual Function ◽  
Wild Type ◽  
Lipid Asymmetry ◽  
Content Type ◽  
Intercellular Spread ◽  
Phospholipid Profile

ABSTRACTThe Vps/VacJ ABC transporter system is proposed to function in maintaining the lipid asymmetry of the outer membrane. Mutations invpsorvacJinShigella flexneriresulted in increased sensitivity to lysis by the detergent sodium dodecyl sulfate (SDS), and thevpsCmutant showed minor differences in its phospholipid profile compared to the wild type.vpsCmutants were unable to form plaques in cultured epithelial cells, but this was not due to a failure to invade, to replicate intracellularly, or to polymerize actin via IcsA for movement within epithelial cells. The addition of the outer membrane phospholipase genepldAon a multicopy plasmid in avpsCorvacJmutant restored its resistance to SDS, suggesting a restoration of lipid asymmetry to the outer membrane. However, thepldAplasmid did not restore the mutant's ability to form plaques in tissue culture cells. Increased PldA levels also failed to restore the mutant's phospholipid profile to that of the wild type. We propose a dual function of the Vps/VacJ ABC transporter system inS. flexneriin both the maintenance of lipid asymmetry in the outer membrane and the intercellular spread of the bacteria between adjacent epithelial cells.

scholarly journals Key Role for DsbA in Cell-to-Cell Spread ofShigella flexneri, Permitting Secretion of Ipa Proteins into Interepithelial Protrusions

2000 ◽  
Vol 68 (11) ◽  
pp. 6449-6456 ◽  
Author(s):  
Jun Yu ◽  
Bryn Edwards-Jones ◽  
Olivier Neyrolles ◽  
J. Simon Kroll
Keyword(s):  
Confocal Microscopy ◽  
Disulfide Bond ◽  
Active Site ◽  
Shigella Flexneri ◽  
Wild Type ◽  
Pathogenic Potential ◽  
Previous Demonstration ◽  
Intercellular Spread ◽  
Cell Spread

ABSTRACT DsbA, a disulfide bond catalyst, is necessary for realization of the pathogenic potential of Shigella flexneri. Sh42, a mutant strain differing from wild-type M90TS solely because it expresses nonfunctional DsbA33G (substitution for 33C at the active site), secreted less IpaB and IpaC than M90TS in response to various stimuli in vitro. A kinetic study demonstrated that Sh42 responded more slowly to Congo red than M90TS. By modulating relative concentrations of functional and nonfunctional DsbA within bacteria, functional enzyme has been shown to be necessary for intercellular spread. By confocal microscopy, M90TS dividing in protrusions was shown to secrete Ipa proteins from the septation furrow, anticipating lysis of protrusions, while Sh42 showed minimal Ipa secretion in this location. In the light of a previous demonstration that DsbA is not necessary for entry of epithelial cells, we conclude that a role in virulence of this disulfide bond catalyst lies in facilitating secretion of Ipa proteins specifically within epithelial protrusions, in turn allowing cell-to-cell spread of S. flexneri.

icsB: a Shigella flexneri virulence gene necessary for the lysis of protrusions during intercellular spread

1992 ◽  
Vol 6 (12) ◽  
pp. 1605-1616 ◽  
Author(s):  
Abdelmounaaïm Allaoui ◽  
Joëlle Mounier ◽  
Marie-Christine Prévost ◽  
Philippe J. Sansonetti ◽  
Claude Parsot
Keyword(s):  
Shigella Flexneri ◽  
Virulence Gene ◽  
Intercellular Spread

scholarly journals Inactivation of DsbA, but Not DsbC and DsbD, Affects the Intracellular Survival and Virulence ofShigella flexneri

1998 ◽  
Vol 66 (8) ◽  
pp. 3909-3917 ◽  
Author(s):  
Jun Yu
Keyword(s):  
Hela Cells ◽  
Shigella Flexneri ◽  
Wild Type ◽  
Electron Microscopy Analysis ◽  
Attenuation Mechanism ◽  
Serotype 5 ◽  
Type Gene ◽  
Intercellular Spread

ABSTRACT In this study, three mutants,dsbA::kan, dsbC-kan, anddsbD-kan, of Shigella flexneri serotype 5 were constructed and characterized to investigate the role of the periplasmic thiol:disulfide oxidoreductases in pathogenicity. In gentamicin protection assays and the Serény test, thedsbA mutant showed reduced virulence while thedsbC and dsbD mutants were similar to the wild type. That inactivation of dsbA was responsible for the reduced virulence was verified by complementation with the cloned wild-type gene in in vitro and in vivo assays. Despite the changed virulence behavior, the dsbA mutant could penetrate HeLa cells 15 min postinfection, consistent with the fact that it actively secretes Ipa proteins upon Congo red induction. Furthermore, thedsbA mutant was able to produce actin comets and protrusions, indicating its capacity for intra- and intercellular spread. However, a kinetic analysis of intracellular growth showed that the dsbA mutant barely grew in HeLa cells during a 4-h infection whereas the wild type had a doubling time of 41 min. Electron microscopy analysis revealed that dsbA mutant bacteria were trapped in protrusion-derived vacuoles surrounded by double membranes, resembling an icsB mutant reported previously. Moreover, the trapped bacteria appeared to be lysed simultaneously with the double membranes, resulting in characteristic empty vacuoles in the host cell cytosol. Thus, the attenuation mechanism for dsbAmutant appears to be more complicated than was previously suggested.

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