Role of Lipooligosaccharide in Opa-Independent Invasion of Neisseria gonorrhoeae into Human Epithelial Cells

Lipooligosaccharide (LOS) has been implicated in the adhesion and invasion of host epithelial cells. We examined the adhesive and invasive abilities of isogenic gonococcal opacity-associated outer membrane protein–negative, pilus-positive (Opa−Pil+) Neisseria gonorrhoeae strains expressing genetically defined LOS. Strain F62 (Opa−Pil+), expressing the lacto-N-neotetraose and the galNac-lacto-N-neotetraose LOS, and its isogenic derivative that expressed only the lacto-N-neotetraose LOS (F62ΔlgtD), adhered to, and invaded, to the same extent the human cervical epidermoid carcinoma cell line, ME180. While the adhesive abilities of Opa−Pil+ isogenic strains that express LOS molecules lacking the lacto-N-neotetraose structure were similar to that seen for F62, their invasive abilities were much lower than the strains expressing lacto-N-neotetraose. Fluorescence microscopy studies showed that the adherence of F62, but not the strains lacking lacto-N-neotetraose, induced the rearrangement of actin filaments under the adherent sites. Electron microscopy studies demonstrated that F62, but not the strains lacking lacto-N-neotetraose, formed extensive and intimate associations with epithelial cell membranes. Thus, in the absence of detectable Opa protein, the lacto-N-neotetraose LOS promotes gonococcal invasion into ME180 cells. The data also suggest that LOS is involved in the mobilization of actin filaments in host cells, and in the formation of a direct interaction between the bacterial outer membrane and the plasma membrane of ME180 cells.

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Global Gene Expression and the Role of Sigma Factors in Neisseria gonorrhoeae in Interactions with Epithelial Cells

Infection and Immunity ◽

10.1128/iai.73.8.4834-4845.2005 ◽

2005 ◽

Vol 73 (8) ◽

pp. 4834-4845 ◽

Cited By ~ 30

Author(s):

Ying Du ◽

Jonathan Lenz ◽

Cindy Grove Arvidson

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Neisseria Gonorrhoeae ◽

Sigma Factor ◽

Dna Microarrays ◽

Environmental Changes ◽

Host Cells ◽

Global Changes ◽

New Host

ABSTRACT Like many bacterial pathogens, Neisseria gonorrhoeae must adapt to environmental changes in order to successfully colonize and proliferate in a new host. Modulation of gene expression in response to environmental signals is an efficient mechanism used by bacteria to achieve this goal. Using DNA microarrays and a tissue culture model for gonococcal infection, we examined global changes in gene expression in N. gonorrhoeae in response to adherence to host cells. Among those genes induced upon adherence to human epithelial cells in culture was rpoH, which encodes a homolog of the heat shock sigma factor, σ32 (RpoH), as well as genes of the RpoH regulon, groEL and groES. Attempts to construct an rpoH null mutant in N. gonorrhoeae were unsuccessful, suggesting that RpoH is essential for viability of N. gonorrhoeae. The extracytoplasmic sigma factor, RpoE (σE), while known to regulate rpoH in other bacteria, was found not to be necessary for the up-regulation of rpoH in gonococci upon adherence to host cells. To examine the role of RpoH in host cell interactions, an N. gonorrhoeae strain conditionally expressing rpoH was constructed. The results of our experiments showed that while induction of rpoH expression is not necessary for adherence of gonococci to epithelial cells, it is important for the subsequent invasion step, as gonococci depleted for rpoH invade cells two- to threefold less efficiently than a wild-type strain. Taken together, these results indicate that σ32, but not σE, is important for the response of gonococci in the initial steps of an infection.

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Aggregatibacter actinomycetemcomitans Leukotoxin Is Delivered to Host Cells in an LFA-1-Indepdendent Manner When Associated with Outer Membrane Vesicles

Toxins ◽

10.3390/toxins10100414 ◽

2018 ◽

Vol 10 (10) ◽

pp. 414 ◽

Cited By ~ 9

Author(s):

Justin Nice ◽

Nataliya Balashova ◽

Scott Kachlany ◽

Evan Koufos ◽

Eric Krueger ◽

...

Keyword(s):

Outer Membrane ◽

Membrane Vesicles ◽

Aggregatibacter Actinomycetemcomitans ◽

Aggressive Periodontitis ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Type I ◽

Outer Membranes ◽

Independent Mechanism ◽

Bacterial Outer Membrane

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, has been associated with localized aggressive periodontitis (LAP). In particular, highly leukotoxic strains of A. actinomycetemcomitans have been more closely associated with this disease, suggesting that LtxA is a key virulence factor for A. actinomycetemcomitans. LtxA is secreted across both the inner and outer membranes via the Type I secretion system, but has also been found to be enriched within outer membrane vesicles (OMVs), derived from the bacterial outer membrane. We have characterized the association of LtxA with OMVs produced by the highly leukotoxic strain, JP2, and investigated the interaction of these OMVs with host cells to understand how LtxA is delivered to host cells in this OMV-associated form. Our results demonstrated that a significant fraction of the secreted LtxA exists in an OMV-associated form. Furthermore, we have discovered that in this OMV-associated form, the toxin is trafficked to host cells by a cholesterol- and receptor-independent mechanism in contrast to the mechanism by which free LtxA is delivered. Because OMV-associated toxin is trafficked to host cells in an entirely different manner than free toxin, this study highlights the importance of studying both free and OMV-associated forms of LtxA to understand A. actinomycetemcomitans virulence.

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OmpD but not OmpC is involved in adherence ofSalmonella entericaserovar Typhimurium to human cells

Canadian Journal of Microbiology ◽

10.1139/w04-056 ◽

2004 ◽

Vol 50 (9) ◽

pp. 719-727 ◽

Cited By ~ 17

Author(s):

Bochiwe Hara-Kaonga ◽

Thomas G Pistole

Keyword(s):

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Human Cells ◽

Host Cells ◽

Intestinal Epithelial ◽

Wild Type ◽

Human Macrophages ◽

Significant Difference ◽

Serovar Typhimurium

Conflicting reports exist regarding the role of porins OmpC and OmpD in infections due to Salmonella enterica serovar Typhimurium. This study investigated the role of these porins in bacterial adherence to human macrophages and intestinal epithelial cells. ompC and ompD mutant strains were created by transposon mutagenesis using P22-mediated transduction of Tn10 and Tn5 insertions, respectively, into wild-type strain 14028. Fluorescein-labeled wild-type and mutant bacteria were incubated with host cells at various bacteria to cell ratios for 1 h at 37 °C and analyzed by flow cytometry. The mean fluorescence intensity of cells with associated wild-type and mutant bacteria was used to estimate the number of bacteria bound per host cell. Adherence was also measured by fluorescence microscopy. Neither assay showed a significant difference in binding of the ompC mutant and wild-type strains to the human cells. In contrast, the ompD mutant exhibited lowered binding to both cell types. Our findings suggest that OmpD but not OmpC is involved in the recognition of Salmonella serovar Typhimurium by human macrophages and intestinal epithelial cells.Key words: Salmonella, adherence, porins, intestinal epithelial cells, macrophage.

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Role of Outer-membrane Proteins and Lipopolysaccharide in Conjugation between Neisseria gonorrhoeae and Neisseria cinerea

Microbiology ◽

10.1099/00221287-134-12-3285 ◽

1988 ◽

Vol 134 (12) ◽

pp. 3285-3294

Author(s):

C. A. GENCO ◽

V. L. CLARK

Keyword(s):

Membrane Proteins ◽

Neisseria Gonorrhoeae ◽

Outer Membrane ◽

Outer Membrane Proteins

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Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts.

The Journal of Cell Biology ◽

10.1083/jcb.135.1.153 ◽

1996 ◽

Vol 135 (1) ◽

pp. 153-167 ◽

Cited By ~ 180

Author(s):

J Peränen ◽

P Auvinen ◽

H Virta ◽

R Wepf ◽

K Simons

Keyword(s):

Epithelial Cells ◽

Membrane Transport ◽

Vesicular Stomatitis Virus ◽

Actin Filaments ◽

Mdck Cells ◽

Basolateral Membrane ◽

Wild Type ◽

Expression Systems ◽

Vesicular Stomatitis

Rab8 is a small Ras-like GTPase that regulates polarized membrane transport to the basolateral membrane in epithelial cells and to the dendrites in neurons. It has recently been demonstrated that fibroblasts sort newly synthesized proteins into two different pathways for delivery to the cell surface that are equivalent to the apical and the basolateral post-Golgi routes in epithelial cells (Yoshimori, T., P. Keller, M.G. Roth, and K. Simons. 1996. J. Cell Biol. 133:247-256). To determine the role of Rab8 in fibroblasts, we used both transient expression systems and stable cell lines expressing mutant or wild-type (wt) Rab8. A dramatic change in cell morphology occurred in BHK cells expressing both the wt Rab8 and the activated form of the GTPase, the Rab8Q67L mutant. These cells formed processes as a result of a reorganization of both their actin filaments and microtubules. Newly synthesized vesicular stomatitis virus G glycoprotein, a basolateral marker protein in MDCK cells, was preferentially delivered into these cell outgrowths. Based on these findings, we propose that Rab8 provides a link between the machinery responsible for the formation of cell protrusions and polarized biosynthetic membrane traffic.

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RpoH Mediates the Expression of Some, but Not All, Genes Induced in Neisseria gonorrhoeae Adherent to Epithelial Cells

Infection and Immunity ◽

10.1128/iai.74.5.2767-2776.2006 ◽

2006 ◽

Vol 74 (5) ◽

pp. 2767-2776 ◽

Cited By ~ 3

Author(s):

Ying Du ◽

Cindy Grove Arvidson

Keyword(s):

Epithelial Cells ◽

Neisseria Gonorrhoeae ◽

Host Cell ◽

Infection Process ◽

Host Cells ◽

Cell Contact ◽

Transmitted Infection ◽

New Host ◽

Sexually Transmitted ◽

Cell Induction

ABSTRACT Neisseria gonorrhoeae (gonococcus [GC]), is highly adapted to the human host, the only known reservoir for gonococcal infection. However, since it is sexually transmitted, infection of a new host likely requires a regulatory response on the part of the gonococcus to respond to this significant change in environment. We previously showed that adherence of gonococci to epithelial cells results in changes of gene expression in the bacteria that presumably prepare them for subsequent steps in the infection process. Expression of the heat shock sigma factor gene, rpoH, was shown to be important for the invasion step, as gonococci depleted for rpoH were reduced in their ability to invade epithelial cells. Here, we show that of the genes induced in adherent gonococci, two are part of the gonococcal RpoH regulon. When RpoH is depleted, expression of these genes is no longer induced by host cell contact, indicating that RpoH is mediating the host cell induction response of these genes. One RpoH-dependent gene, NGO0376, is shown to be important for invasion of epithelial cells, consistent with earlier observations that RpoH is necessary for this step of infection. Two genes, NGO1684 and NGO0340, while greatly induced by host cell contact, were found to be RpoH independent, indicating that more than one regulator is involved in the response to host cell contact. Furthermore, NGO0340, but not NGO1684, was shown to be important for both adherence and invasion of epithelial cells, suggesting a complex regulatory network in the response of gonococci to contact with host cells.

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Receptor-mediated endocytosis of Neisseria gonorrhoeae into primary human urethral epithelial cells: the role of the asialoglycoprotein receptor

Molecular Microbiology ◽

10.1046/j.1365-2958.2001.02666.x ◽

2008 ◽

Vol 42 (3) ◽

pp. 659-672 ◽

Cited By ~ 75

Author(s):

H. A. Harvey ◽

M. P. Jennings ◽

C. A. Campbell ◽

R. Williams ◽

M. A. Apicella

Keyword(s):

Epithelial Cells ◽

Neisseria Gonorrhoeae ◽

Asialoglycoprotein Receptor ◽

Receptor Mediated Endocytosis

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Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

Molecular Biology of the Cell ◽

10.1091/mbc.e10-12-0939 ◽

2011 ◽

Vol 22 (8) ◽

pp. 1290-1299 ◽

Cited By ~ 42

Author(s):

Simren Mehta ◽

L. David Sibley

Keyword(s):

Toxoplasma Gondii ◽

Actin Filaments ◽

Gliding Motility ◽

Conditional Knockout ◽

Host Cells ◽

Actin Binding ◽

Actin Depolymerizing Factor

Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ∼98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer–sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.

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Neisseria gonorrhoeae Porin P1.B Induces Endosome Exocytosis and a Redistribution of Lamp1 to the Plasma Membrane

Infection and Immunity ◽

10.1128/iai.70.11.5965-5971.2002 ◽

2002 ◽

Vol 70 (11) ◽

pp. 5965-5971 ◽

Cited By ~ 16

Author(s):

Patricia Ayala ◽

Brandi Vasquez ◽

Lee Wetzler ◽

Magdalene So

Keyword(s):

Plasma Membrane ◽

Epithelial Cells ◽

Cell Surface ◽

Neisseria Gonorrhoeae ◽

Bacterial Infection ◽

Immunoglobulin A ◽

Late Endosomes ◽

A Cell ◽

Iga Protease

ABSTRACT The immunoglobulin A (IgA) protease secreted by pathogenic Neisseria spp. cleaves Lamp1, thereby altering lysosomes in a cell and promoting bacterial intracellular survival. We sought to determine how the IgA protease gains access to cellular Lamp1 in order to better understand the role of this cleavage event in bacterial infection. In a previous report, we demonstrated that the pilus-induced Ca2+ transient triggers lysosome exocytosis in human epithelial cells. This, in turn, increases the level of Lamp1 at the plasma membrane, where it can be cleaved by IgA protease. Here, we show that porin also induces a Ca2+ flux in epithelial cells. This transient is similar in nature to that observed in phagocytes exposed to porin. In contrast to the pilus-induced Ca2+ transient, the porin-induced event does not trigger lysosome exocytosis. Instead, it stimulates exocytosis of early and late endosomes and increases Lamp1 on the cell surface. These results indicate that Neisseria pili and porin perturb Lamp1 trafficking in epithelial cells by triggering separate and distinct Ca2+-dependent exocytic events, bringing Lamp1 to the cell surface, where it can be cleaved by IgA protease.

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