scholarly journals 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

2015 ◽  
Vol 22 (12) ◽  
pp. 1255-1268 ◽  
Author(s):  
Bethany L. Tiner ◽  
Jian Sha ◽  
Duraisamy Ponnusamy ◽  
Wallace B. Baze ◽  
Eric C. Fitts ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Cell Mediated Immunity ◽  
Interleukin 17 ◽  
Triple Mutant ◽  
Pneumonic Plague ◽  
Content Type ◽  
Virulence Potential ◽  
Genes Encoding ◽  
Subsequent Exposure ◽  
Vaccine Potential

ABSTRACTEarlier, we showed that the ΔlppΔmsbBΔailtriple mutant ofYersinia pestisCO92 with deleted genes encoding Braun lipoprotein (Lpp), an acyltransferase (MsbB), and the attachment invasion locus (Ail), respectively, was avirulent in a mouse model of pneumonic plague. In this study, we further evaluated the immunogenic potential of the ΔlppΔmsbBΔailtriple mutant and its derivative by different routes of vaccination. Mice were immunized via the subcutaneous (s.c.) or the intramuscular (i.m.) route with two doses (2 × 106CFU/dose) of the above-mentioned triple mutant with 100% survivability of the animals. Upon subsequent pneumonic challenge with 70 to 92 50% lethal doses (LD50) of wild-type (WT) strain CO92, all of the mice survived when immunization occurred by the i.m. route. Since Ail has virulence and immunogenic potential, a mutated version of Ail devoid of its virulence properties was created, and the genetically modifiedailreplaced the nativeailgene on the chromosome of the ΔlppΔmsbBdouble mutant, creating a ΔlppΔmsbB::ailL2vaccine strain. This newly generated mutant was attenuated similarly to the ΔlppΔmsbBΔailtriple mutant when administered by the i.m. route and provided 100% protection to animals against subsequent pneumonic challenge. Not only were the two above-mentioned mutants cleared rapidly from the initial i.m. site of injection in animals with no histopathological lesions, the immunized mice did not exhibit any disease symptoms during immunization or after subsequent exposure to WT CO92. These two mutants triggered balanced Th1- and Th2-based antibody responses and cell-mediated immunity. A substantial increase in interleukin-17 (IL-17) from the T cells of vaccinated mice, a cytokine of the Th17 cells, further augmented their vaccine potential. Thus, the ΔlppΔmsbBΔailand ΔlppΔmsbB::ailL2mutants represent excellent vaccine candidates for plague, with the latter mutant still retaining Ail immunogenicity but with a much diminished virulence potential.

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

2015 ◽  
Vol 83 (4) ◽  
pp. 1318-1338 ◽  
Author(s):  
Bethany L. Tiner ◽  
Jian Sha ◽  
Michelle L. Kirtley ◽  
Tatiana E. Erova ◽  
Vsevolod L. Popov ◽  
...  
Keyword(s):  
Mouse Model ◽  
Yersinia Pestis ◽  
Double Mutant ◽  
Lethal Dose ◽  
Bactericidal Effect ◽  
Alveolar Epithelial Cells ◽  
Triple Mutant ◽  
Pneumonic Plague ◽  
Content Type

Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuatedYersinia pestisCO92 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 theailgene (encoding theattachment-invasionlocus) from wild-type (WT) strain CO92 or itslppsingle and ΔlppΔmsbBdouble mutants. While the Δailsingle mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the ΔlppΔaildouble mutant and the ΔlppΔmsbBΔailtriple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD50) equivalent to 6,800 LD50s of WT CO92. The mutant-infected animals developed balanced TH1- and TH2-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 LD50s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time byin vivoimaging. 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 theailgene from the ΔlppΔmsbBdouble mutant severely attenuatedY. pestisCO92 to evoke pneumonic plague in a mouse model while retaining the required immunogenicity needed for subsequent protection against infection.

scholarly journals High-Throughput, Signature-Tagged Mutagenic Approach To Identify Novel Virulence Factors of Yersinia pestis CO92 in a Mouse Model of Infection

2015 ◽  
Vol 83 (5) ◽  
pp. 2065-2081 ◽  
Author(s):  
Duraisamy Ponnusamy ◽  
Eric C. Fitts ◽  
Jian Sha ◽  
Tatiana E. Erova ◽  
Elena V. Kozlova ◽  
...  
Keyword(s):  
Mouse Model ◽  
Yersinia Pestis ◽  
Virulence Factors ◽  
High Throughput ◽  
Molecular Mechanisms ◽  
Sugar Transport ◽  
Type Vi Secretion System ◽  
Pneumonic Plague ◽  
Content Type ◽  
Genes Encoding

The identification of new virulence factors inYersinia pestisand understanding their molecular mechanisms during an infection process are necessary in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a high-throughput, signature-tagged mutagenic approach, we created 5,088 mutants ofY. pestisstrain CO92 and screened them in a mouse model of pneumonic plague at a dose equivalent to 5 50% lethal doses (LD50) of wild-type (WT) CO92. From this screen, we obtained 118 clones showing impairment in disseminating to the spleen, based on hybridization of input versus output DNA from mutant pools with 53 unique signature tags. In the subsequent screen, 20/118 mutants exhibited attenuation at 8 LD50when tested in a mouse model of bubonic plague, with infection by 10/20 of the aforementioned mutants resulting in 40% or higher survival rates at an infectious dose of 40 LD50. Upon sequencing, six of the attenuated mutants were found to carry interruptions in genes encoding hypothetical proteins or proteins with putative functions. Mutants with in-frame deletion mutations of two of the genes identified from the screen, namely,rbsA, which codes for a putative sugar transport system ATP-binding protein, andvasK, a component of the type VI secretion system, were also found to exhibit some attenuation at 11 or 12 LD50in a mouse model of pneumonic plague. Likewise, among the remaining 18 signature-tagged mutants, 9 were also attenuated (40 to 100%) at 12 LD50in a pneumonic plague mouse model. Previously, we found that deleting genes encoding Braun lipoprotein (Lpp) and acyltransferase (MsbB), the latter of which modifies lipopolysaccharide function, reduced the virulence ofY. pestisCO92 in mouse models of bubonic and pneumonic plague. Deletion ofrbsAandvasKgenes from either the Δlppsingle or the ΔlppΔmsbBdouble mutant augmented the attenuation to provide 90 to 100% survivability to mice in a pneumonic plague model at 20 to 50 LD50. The mice infected with the ΔlppΔmsbBΔrbsAtriple mutant at 50 LD50were 90% protected upon subsequent challenge with 12 LD50of WT CO92, suggesting that this mutant or others carrying combinational deletions of genes identified through our screen could potentially be further tested and developed into a live attenuated plague vaccine(s).

scholarly journals Inhibition of Neutrophil Primary Granule Release during Yersinia pestis Pulmonary Infection

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Kara R. Eichelberger ◽  
Grant S. Jones ◽  
William E. Goldman
Keyword(s):  
Yersinia Pestis ◽  
Critical Role ◽  
Immune Defense ◽  
Calcium Flux ◽  
Human Neutrophils ◽  
Pneumonic Plague ◽  
Content Type ◽  
Neutrophil Degranulation ◽  
Primary Granule ◽  
Granule Release

ABSTRACT Inhalation of Yersinia pestis causes primary pneumonic plague, the most severe manifestation of plague that is characterized by a dramatic neutrophil influx to the lungs. Neutrophils are ineffective during primary pneumonic plague, failing to control Y. pestis growth in the airways. However, the mechanisms by which Y. pestis resists neutrophil killing are incompletely understood. Here, we show that Y. pestis inhibits neutrophil degranulation, an important line of host innate immune defense. We observed that neutrophils from the lungs of mice infected intranasally with Y. pestis fail to release primary granules throughout the course of disease. Using a type III secretion system (T3SS) injection reporter strain, we determined that Y. pestis directly inhibits neutrophil granule release by a T3SS-dependent mechanism. Combinatorial mutant analysis revealed that a Y. pestis strain lacking both effectors YopE and YopH did not inhibit primary granule release and is killed by neutrophils both in vivo and in vitro. Similarly, Y. pestis strains injecting only YopE or YopH are able to inhibit the majority of primary granule release from human neutrophils. We determined that YopE and YopH block Rac2 activation and calcium flux, respectively, to inhibit neutrophil primary granule release in isolated human neutrophils. These results demonstrate that Y. pestis coordinates the inhibition of neutrophil primary granule release through the activities of two distinct effectors, and this inhibition promotes Y. pestis survival during primary pneumonic plague. IMPORTANCE Yersinia pestis is the causative agent of plague and is one of the deadliest human pathogens. The pneumonic form of Y. pestis infection has played a critical role in the severity of both historical and modern plague outbreaks, yet the host-pathogen interactions that govern the lethality of Yersinia pestis pulmonary infections are incompletely understood. Here, we report that Yersinia pestis inhibits neutrophil degranulation during infection, rendering neutrophils ineffective and allowing unrestricted growth of Y. pestis in the lungs. This coordinated inhibition of granule release not only demonstrates the pathogenic benefit of “silencing” lung neutrophils but also reveals specific host processes and pathways that could be manipulated to reduce the severity of primary pneumonic plague.

scholarly journals Combinatorial Viral Vector-Based and Live Attenuated Vaccines without an Adjuvant to Generate Broader Immune Responses to Effectively Combat Pneumonic Plague

mBio ◽  
2021 ◽  
Author(s):  
Paul B. Kilgore ◽  
Jian Sha ◽  
Emily K. Hendrix ◽  
Vladimir L. Motin ◽  
Ashok K. Chopra
Keyword(s):  
Immune Responses ◽  
Yersinia Pestis ◽  
Causative Agent ◽  
Viral Vector ◽  
Human Pathogen ◽  
Pneumonic Plague ◽  
Content Type ◽  
Live Attenuated Vaccines ◽  
Tier 1

Yersinia pestis , the causative agent of plague, is a Tier-1 select agent and a reemerging human pathogen. A 2017 outbreak in Madagascar with >75% of cases being pneumonic and 8.6% causalities emphasized the importance of the disease.

scholarly journals In Vivo Transcriptional Profiling of Yersinia pestis Reveals a Novel Bacterial Mediator of Pulmonary Inflammation

mBio ◽  
2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Roger D. Pechous ◽  
Christopher A. Broberg ◽  
Nikolas M. Stasulli ◽  
Virginia L. Miller ◽  
William E. Goldman
Keyword(s):  
Yersinia Pestis ◽  
Inflammatory Responses ◽  
Severe Pneumonia ◽  
Sudden Onset ◽  
Host Factors ◽  
Respiratory Pathogen ◽  
Bacterial Survival ◽  
Pneumonic Plague ◽  
Content Type

ABSTRACTInhalation ofYersinia pestisresults in primary pneumonic plague, a highly lethal and rapidly progressing necrotizing pneumonia. The disease begins with a period of extensive bacterial replication in the absence of disease symptoms, followed by the sudden onset of inflammatory responses that ultimately prove fatal. Very little is known about the bacterial and host factors that contribute to the rapid biphasic progression of pneumonic plague. In this work, we analyzed thein vivotranscription kinetics of 288 bacterial open reading frames previously shown by microarray analysis to be dynamically regulated in the lung. Using this approach combined with bacterial genetics, we were able to identify five Y. pestis genes that contribute to the development of pneumonic plague. Deletion of one of these genes,ybtX, did not alter bacterial survival but attenuated host inflammatory responses during late-stage disease. Deletion ofybtXin another lethal respiratory pathogen,Klebsiella pneumoniae, also resulted in diminished host inflammation during infection. Thus, ourin vivotranscriptional screen has identified an important inflammatory mediator that is common to two Gram-negative bacterial pathogens that cause severe pneumonia.IMPORTANCEYersinia pestis is responsible for at least three major pandemics, most notably the Black Death of the Middle Ages. Due to its pandemic potential, ease of dissemination by aerosolization, and a history of its weaponization, Y. pestis is categorized by the Centers for Disease Control and Prevention as a tier 1 select agent most likely to be used as a biological weapon. To date, there is no licensed vaccine against Y. pestis. Importantly, an early “silent” phase followed by the rapid onset of nondescript influenza-like symptoms makes timely treatment of pneumonic plague difficult. A more detailed understanding of the bacterial and host factors that contribute to pathogenesis is essential to understanding the progression of pneumonic plague and developing or enhancing treatment options.

scholarly journals LcrV Delivered via Type III Secretion System of Live Attenuated Yersinia pseudotuberculosis Enhances Immunogenicity against Pneumonic Plague

2014 ◽  
Vol 82 (10) ◽  
pp. 4390-4404 ◽  
Author(s):  
Wei Sun ◽  
Shilpa Sanapala ◽  
Jeremy C. Henderson ◽  
Shandiin Sam ◽  
Joseph Olinzock ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Yersinia Pseudotuberculosis ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Secretory Iga ◽  
Interleukin 17 ◽  
Necrosis Factor Alpha ◽  
Pneumonic Plague ◽  
Content Type ◽  
Type Iii

ABSTRACTHere, we constructed aYersinia pseudotuberculosismutant strain with arabinose-dependent regulated and delayed shutoff ofcrpexpression (araCPBADcrp) and replacement of themsbBgene with theEscherichia colimsbBgene to attenuate it. Then, we inserted theasdmutation into this construction to form χ10057 [Δasd-206ΔmsbB868::PmsbBmsbB(EC)ΔPcrp21::TTaraCPBADcrp] for use with a balanced-lethal Asd-positive (Asd+) plasmid to facilitate antigen synthesis. A hybrid protein composed of YopE (amino acids [aa]1 to 138) fused with full-length LcrV (YopENt138-LcrV) was synthesized in χ10057 harboring an Asd+plasmid (pYA5199,yopENt138-lcrV) and could be secreted through a type III secretion system (T3SS)in vitroandin vivo. Animal studies indicated that mice orally immunized with χ10057(pYA5199) developed titers of IgG response to whole-cell lysates ofY. pestis(YpL) and subunit LcrV similar to those seen with χ10057(pYA3332) (χ10057 plus an empty plasmid). However, only immunization of mice with χ10057(pYA5199) resulted in a significant secretory IgA response to LcrV. χ10057(pYA5199) induced a higher level of protection (80% survival) against intranasal (i.n.) challenge with ∼240 median lethal doses (LD50) (2.4 × 104CFU) ofY. pestisKIM6+(pCD1Ap) than χ10057(pYA3332) (40% survival). Splenocytes from mice vaccinated with χ10057(pYA5199) produced significant levels of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-17 (IL-17) after restimulation with LcrV and YpL antigens. Our results suggest that it is possible to use an attenuatedY. pseudotuberculosisstrain delivering the LcrV antigen via the T3SS as a potential vaccine candidate against pneumonic plague.

scholarly journals Borrelia Host Adaptation Protein (BadP) Is Required for the Colonization of a Mammalian Host by the Agent of Lyme Disease

2018 ◽  
Vol 86 (7) ◽  
Author(s):  
Trever C. Smith ◽  
Sarah M. Helm ◽  
Yue Chen ◽  
Ying-Han Lin ◽  
S. L. Rajasekhar Karna ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lyme Disease ◽  
Immunoblot Analysis ◽  
Growth Defect ◽  
Mammalian Host ◽  
Content Type ◽  
Magnesium Chelatase ◽  
Virulence Potential ◽  
Genes Encoding

ABSTRACT Borrelia burgdorferi , the agent of Lyme disease (LD), uses host-derived signals to modulate gene expression during the vector and mammalian phases of infection. Microarray analysis of mutants lacking the B orrelia host ad aptation r egulator (BadR) revealed the downregulation of genes encoding enzymes whose role in the pathophysiology of B. burgdorferi is unknown. Immunoblot analysis of the badR mutants confirmed reduced levels of these enzymes, and one of these enzymes, encoded by bb0086 , shares homology to prokaryotic magnesium chelatase and Lon-type proteases. The BB0086 levels in B. burgdorferi were higher under conditions mimicking those in fed ticks. Mutants lacking bb0086 had no apparent in vitro growth defect but were incapable of colonizing immunocompetent C3H/HeN or immunodeficient SCID mice. Immunoblot analysis revealed reduced levels of proteins critical for the adaptation of B. burgdorferi to the mammalian host, such as OspC, DbpA, and BBK32. Both RpoS and BosR, key regulators of gene expression in B. burgdorferi , were downregulated in the bb0086 mutants. Therefore, we designated BB0086 the B orrelia host ad aptation p rotein (BadP). Unlike badP mutants, the control strains established infection in C3H/HeN mice at 4 days postinfection, indicating an early colonization defect in mutants due to reduced levels of the lipoproteins/regulators critical for initial stages of infection. However, badP mutants survived within dialysis membrane chambers (DMCs) implanted within the rat peritoneal cavity but, unlike the control strains, did not display complete switching of OspA to OspC, suggesting incomplete adaptation to the mammalian phase of infection. These findings have opened a novel regulatory mechanism which impacts the virulence potential of B . burgdorferi .

scholarly journals A Bacteriophage T4 Nanoparticle-Based Dual Vaccine against Anthrax and Plague

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Pan Tao ◽  
Marthandan Mahalingam ◽  
Jingen Zhu ◽  
Mahtab Moayeri ◽  
Jian Sha ◽  
...  
Keyword(s):  
Immune Responses ◽  
Bacillus Anthracis ◽  
Yersinia Pestis ◽  
Pathogenic Bacteria ◽  
Bacteriophage T4 ◽  
The United States ◽  
Complete Protection ◽  
Pneumonic Plague ◽  
Content Type ◽  
Phage T4

ABSTRACT Bacillus anthracis and Yersinia pestis, the causative agents of anthrax and plague, respectively, are two of the deadliest pathogenic bacteria that have been used as biological warfare agents. Although Biothrax is a licensed vaccine against anthrax, no Food and Drug Administration-approved vaccine exists for plague. Here, we report the development of a dual anthrax-plague nanoparticle vaccine employing bacteriophage (phage) T4 as a platform. Using an in vitro assembly system, the 120- by 86-nm heads (capsids) of phage T4 were arrayed with anthrax and plague antigens fused to the small outer capsid protein Soc (9 kDa). The antigens included the anthrax protective antigen (PA) (83 kDa) and the mutated (mut) capsular antigen F1 and the low-calcium-response V antigen of the type 3 secretion system from Y. pestis (F1mutV) (56 kDa). These viral nanoparticles elicited robust anthrax- and plague-specific immune responses and provided complete protection against inhalational anthrax and/or pneumonic plague in three animal challenge models, namely, mice, rats, and rabbits. Protection was demonstrated even when the animals were simultaneously challenged with lethal doses of both anthrax lethal toxin and Y. pestis CO92 bacteria. Unlike the traditional subunit vaccines, the phage T4 vaccine uses a highly stable nanoparticle scaffold, provides multivalency, requires no adjuvant, and elicits broad T-helper 1 and 2 immune responses that are essential for complete clearance of bacteria during infection. Therefore, phage T4 is a unique nanoparticle platform to formulate multivalent vaccines against high-risk pathogens for national preparedness against potential bioterror attacks and emerging infections. IMPORTANCE Following the deadly anthrax attacks of 2001, the Centers for Disease Control and Prevention (CDC) determined that Bacillus anthracis and Yersinia pestis that cause anthrax and plague, respectively, are two Tier 1 select agents that pose the greatest threat to the national security of the United States. Both cause rapid death, in 3 to 6 days, of exposed individuals. We engineered a virus nanoparticle vaccine using bacteriophage T4 by incorporating key antigens of both B. anthracis and Y. pestis into one formulation. Two doses of this vaccine provided complete protection against both inhalational anthrax and pneumonic plague in animal models. This dual anthrax-plague vaccine is a strong candidate for stockpiling against a potential bioterror attack involving either one or both of these biothreat agents. Further, our results establish the T4 nanoparticle as a novel platform to develop multivalent vaccines against pathogens of high public health significance.

scholarly journals Identification and Characterization of Cronobacter Iron Acquisition Systems

2012 ◽  
Vol 78 (17) ◽  
pp. 6035-6050 ◽  
Author(s):  
C. J. Grim ◽  
M. H. Kothary ◽  
G. Gopinath ◽  
K. G. Jarvis ◽  
J. Jean-Gilles Beaubrun ◽  
...  
Keyword(s):  
Plant Pathogens ◽  
Iron Acquisition ◽  
Natural Habitat ◽  
Clinical Samples ◽  
Powdered Infant Formula ◽  
Bacterial Strains ◽  
Emerging Pathogens ◽  
Content Type ◽  
Virulence Potential ◽  
Genes Encoding

ABSTRACTCronobacterspp. are emerging pathogens that cause severe infantile meningitis, septicemia, or necrotizing enterocolitis. Contaminated powdered infant formula has been implicated as the source ofCronobacterspp. in most cases, but questions still remain regarding the natural habitat and virulence potential for each strain. The iron acquisition systems in 231Cronobacterstrains isolated from different sources were identified and characterized. AllCronobacterspp. have both the Feo and Efe systems for acquisition of ferrous iron, and all plasmid-harboring strains (98%) have the aerobactin-like siderophore, cronobactin, for transport of ferric iron. AllCronobacterspp. have the genes encoding an enterobactin-like siderophore, although it was not functional under the conditions tested. Furthermore, allCronobacterspp. have genes encoding five receptors for heterologous siderophores. A ferric dicitrate transport system (fecsystem) is encoded specifically by a subset ofCronobacter sakazakiiandC. malonaticusstrains, of which a high percentage were isolated from clinical samples. Phylogenetic analysis confirmed that thefecsystem is most closely related to orthologous genes present in human-pathogenic bacterial strains. Moreover, all strains ofC. dublinensisandC. muytjensiiencode two receptors, FcuA and Fct, for heterologous siderophores produced by plant pathogens. Identification of putative Fur boxes and expression of the genes under iron-depleted conditions revealed which genes and operons are components of the Fur regulon. Taken together, these results support the proposition thatC. sakazakiiandC. malonaticusmay be more associated with the human host andC. dublinensisandC. muytjensiiwith plants.

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