Ail Proteins of Yersinia pestis and Y. pseudotuberculosis Have Different Cell Binding and Invasion Activities

Tsang, Tiffany; Wiese, Jeffrey S.; Felek, Suleyman; Kronshage, Malte; Krukonis, Eric S.

doi:10.1371/journal.pone.0083621

Cited by 30 publications

(28 citation statements)

References 48 publications

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“…Indeed loops 1 and 3 of Ail in Y. pestis are important for adherence and invasion, with up to 50% reduction in the abovementioned phenotypes when these two loops were mutated (65). However, similar levels of adherence and invasion of the ⌬lpp ⌬msbB::ailL2 and the ⌬lpp ⌬msbB ⌬ail triple mutants in HeLa cells signified the important role of loop 2 in Ail-associated virulence phenotypes and also suggested that a conformational association between various loops of Ail might be necessary for efficient bacterial adherence to and invasion of the host cells.…”

Section: Discussionmentioning

confidence: 99%

Intramuscular Immunization of Mice with a Live-Attenuated Triple Mutant of Yersinia pestis CO92 Induces Robust Humoral and Cell-Mediated Immunity To Completely Protect Animals against Pneumonic Plague

Tiner

Sha

Ponnusamy

et al. 2015

Clin. Vaccine Immunol.

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Y ersinia pestis is the causative agent of plague (1), and there has been a rise in the number of plague cases globally in recent years possibly due to climate changes and shifting of the rodent carrier range (2). The organism is classified as a tier 1 select agent (3-5), and the progression of septicemic and pneumonic forms of plague is very rapidly fatal after the first appearance of symptoms (4, 6-8). Alarmingly, antibiotic-resistant strains of Y. pestis have been isolated from plague patients and also have been engineered for bioweaponization (4). Therefore, vaccination is the optimal strategy for human protection against this deadly disease; however, there are currently no Food and Drug Administration (FDA)-licensed plague vaccines available in the United States (9-11).Although a heat-killed plague vaccine composed of the Y. pestis 195/P strain was in use in the United States until 1999, the production of this vaccine was discontinued because of its effectiveness only against the bubonic plague and not the pneumonic form and also because it was highly reactogenic in humans (12, 13). Various live-attenuated Y. pestis EV76 vaccine strains, which lack the pigmentation locus (pgm) required for iron acquisition, provide protection against bubonic and pneumonic plague and are being used in some parts of the world where plague is endemic (9). However, these EV76-based vaccines are not genetically uniform and are also highly reactogenic (14); hence, they do not meet the standards for FDA approval. In addition, the ⌬pgm mutants of Y. pestis (e.g., the KIM/D27 strain) may not be safe because of a reported case of fatal infection in an individual with hemochromatosis (15,16).In an effort to search for a new live-attenuated plague vaccine, we recently constructed a ⌬lpp ⌬msbB ⌬ail triple mutant, with deleted genes encoding Braun lipoprotein (Lpp), an acetyltransferase (MsbB), and the attachment invasion locus (Ail) (17). Lpp

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Section: Discussionmentioning

confidence: 99%

Intramuscular Immunization of Mice with a Live-Attenuated Triple Mutant of Yersinia pestis CO92 Induces Robust Humoral and Cell-Mediated Immunity To Completely Protect Animals against Pneumonic Plague

Tiner

Sha

Ponnusamy

et al. 2015

Clin. Vaccine Immunol.

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“…However, Ail is the most critical adhesion molecule, followed by Pla and then Psa (75). We have shown that the levels of Pla and F1 as well as the enzymatic activity of Pla were not affected in the ⌬lpp ⌬msbB ⌬ail triple mutant ( Fig.…”

Section: Discussionmentioning

confidence: 77%

“…Although Y. pestis is a facultative intracellular pathogen (2), its ability to invade epithelial cells has been reported only recently (33,44,61,75). Ail is a major mediator responsible for the adherence and invasion of Y. pestis KIM strains or when the ail gene is overexpressed in nonadherent and noninvasive E. coli strains in human epithelial cells of cervical origin (HeLa and Hep-2) (33,34,44,45).…”

Section: Discussionmentioning

confidence: 99%

“…Ail is a major mediator responsible for the adherence and invasion of Y. pestis KIM strains or when the ail gene is overexpressed in nonadherent and noninvasive E. coli strains in human epithelial cells of cervical origin (HeLa and Hep-2) (33,34,44,45). Likewise, mutated versions of the ail gene from Y. pestis, when expressed and produced in E. coli, exhibited phenotypes of decreased adherence and invasion in HeLa cells, compared to E. coli strains expressing the nonmutated ail gene (75). We provided the first evidence that Ail of Y. pestis has a role in adherence to and invasion of the human alveolar A549 epithelial cell line (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Combinational Deletion of Three Membrane Protein-Encoding Genes Highly Attenuates Yersinia pestis while Retaining Immunogenicity in a Mouse Model of Pneumonic Plague

et al. 2015

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f Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and ⌬lpp ⌬msbB double mutants. While the ⌬ail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the ⌬lpp ⌬ail double mutant and the ⌬lpp ⌬msbB ⌬ail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD 50 ) equivalent to 6,800 LD 50 s of WT CO92. The mutant-infected animals developed balanced T H 1-and T H 2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD 50 s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant's sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the ⌬lpp ⌬msbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a mouse model while retaining the required immunogenicity needed for subsequent protection against infection. P athogenic yersiniae lead to two types of diseases: yersiniosis (typified by gastroenteritis caused by Yersinia enterocolitica and Y. pseudotuberculosis) (1) and plague (evoked by Y. pestis) (2, 3). Y. pestis has evolved from Y. pseudotuberculosis within the last 20,000 years by acquiring additional plasmids and pathogenicity islands as well as by deactivating some genes (4-6). This evolutionary adaptation allowed the plague bacterium to maintain a dual life-style in fleas and rodents/mammals and conferred the ability to survive in the blood instead of the intestine (3). Plague manifests itself in three forms: bubonic (acquired from an infected rodent through a flea bite), pneumonic (acquired either directly by aerosol transmission from an infected host's lungs through respiratory droplets or secondarily from bubonic plague), and septicemic (severe bacteremia either directly due to a flea bite or subsequent to bubonic or pneumonic plague) (2). T...

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“…We have previously characterized the different functions of Y. pestis Ail, including adhesion to host cells, binding to purified Fn and Ln, and facilitation of Yop delivery (1,8,(13)(14)(15)32). These previous studies led us to identify the minimal binding site for Ail on Fn, a stretch of just 180 amino acids in the 250-kDa Fn protein molecule.…”

Section: Discussionmentioning

confidence: 99%

Defining the Ail Ligand-Binding Surface: Hydrophobic Residues in Two Extracellular Loops Mediate Cell and Extracellular Matrix Binding To Facilitate Yop Delivery

et al. 2017

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Yersinia pestis, the causative agent of plague, binds host cells to deliver cytotoxic Yop proteins into the cytoplasm that prevent phagocytosis and generation of proinflammatory cytokines. Ail is an eight-stranded β-barrel outer membrane protein with four extracellular loops that mediates cell binding and resistance to human serum. Following the deletion of each of the four extracellular loops that potentially interact with host cells, the Ail-Δloop 2 and Ail-Δloop 3 mutant proteins had no cell-binding activity while Ail-Δloop 4 maintained cell binding (the Ail-Δloop 1 protein was unstable). Using the codon mutagenesis scheme SWIM (selection without isolation of mutants), we identified individual residues in loops 1, 2, and 3 that contribute to host cell binding. While several residues contributed to the binding of host cells and purified fibronectin and laminin, as well as Yop delivery, three mutations, F80A (loop 2), S128A (loop 3), and F130A (loop 3), produced particularly severe defects in cell binding. Combining these mutations led to an even greater reduction in cell binding and severely impaired Yop delivery with only a slight defect in serum resistance. These findings demonstrate that Y. pestis Ail uses multiple extracellular loops to interact with substrates important for adhesion via polyvalent hydrophobic interactions.

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Ail Proteins of Yersinia pestis and Y. pseudotuberculosis Have Different Cell Binding and Invasion Activities

Cited by 30 publications

References 48 publications

Intramuscular Immunization of Mice with a Live-Attenuated Triple Mutant of Yersinia pestis CO92 Induces Robust Humoral and Cell-Mediated Immunity To Completely Protect Animals against Pneumonic Plague

Intramuscular Immunization of Mice with a Live-Attenuated Triple Mutant of Yersinia pestis CO92 Induces Robust Humoral and Cell-Mediated Immunity To Completely Protect Animals against Pneumonic Plague

Combinational Deletion of Three Membrane Protein-Encoding Genes Highly Attenuates Yersinia pestis while Retaining Immunogenicity in a Mouse Model of Pneumonic Plague

Defining the Ail Ligand-Binding Surface: Hydrophobic Residues in Two Extracellular Loops Mediate Cell and Extracellular Matrix Binding To Facilitate Yop Delivery

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