Targeted Knockout of the Rickettsia rickettsii OmpA Surface Antigen Does Not Diminish Virulence in a Mammalian Model System

ABSTRACTStrains ofRickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF), differ dramatically in virulence despite >99% genetic homology. Spotted fever group (SFG) rickettsiae produce two immunodominant outer membrane proteins, rickettsial OmpA (rOmpA) and rOmpB, which are conserved throughout the SFG and thought to be fundamental to pathogenesis. rOmpA is present in all virulent strains ofR. rickettsiibut is not produced in the only documented avirulent strain, Iowa, due to a premature stop codon. Here we report the creation of an isogenicompAmutant in the highly virulent strain Sheila Smith by insertion of intronic RNA to create a premature stop codon 312 bp downstream of the 6,747-bp open reading frame initiation site (int312). Targeted insertion was accomplished using an LtrA group II intron retrohoming system. Growth and entry rates of Sheila SmithompA::int312 in Vero cells remained comparable to those of the wild type. Virulence was assessed in a guinea pig model by challenge with 100 PFU of eitherompA::int312 Sheila Smith or the wild type, but no significant difference in either fever peak (40.5°C) or duration (8 days) were shown between the wild type and the knockout. The ability to disrupt genes in a site-specific manner using an LtrA group II intron system provides an important new tool for evaluation of potential virulence determinants in rickettsial disease research.IMPORTANCER. rickettsiirOmpA is an immunodominant outer membrane autotransporter conserved in the spotted fever group. Previous studies and genomic comparisons suggest that rOmpA is involved in adhesion and may be critical for virulence. Little information is available for rickettsial virulence factors in an isogenic background, as limited systems for targeted gene disruption are currently available. Here we describe the creation of an rOmpA knockout by insertion of a premature stop codon into the 5′ end of the open reading frame using a group II intron system. An isogenic rOmpA knockout mutation in the highly virulent Sheila Smith strain did not cause attenuation in a guinea pig model of infection, and no altered phenotype was observed in cell culture. We conclude that rOmpA is not critical for virulence in a guinea pig model but may play a role in survival or transmission from the tick vector.

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The guinea pig model for tick-borne spotted fever rickettsioses: A second look

Ticks and Tick-borne Diseases ◽

10.1016/j.ttbdis.2020.101538 ◽

2020 ◽

Vol 11 (6) ◽

pp. 101538

Author(s):

John V. Stokes ◽

David H. Walker ◽

Andrea S. Varela-Stokes

Keyword(s):

Guinea Pig ◽

Spotted Fever ◽

Pig Model ◽

Second Look ◽

Guinea Pig Model

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Beyond the IFA: Revisiting the ELISA as a More Sensitive, Objective, and Quantitative Evaluation of Spotted Fever Group Rickettsia Exposure

Pathogens ◽

10.3390/pathogens10020088 ◽

2021 ◽

Vol 10 (2) ◽

pp. 88

Author(s):

Navatha Alugubelly ◽

John V. Stokes ◽

Claire E. Cross ◽

Anne-Marie L. Ross ◽

Anna E. Crawford ◽

...

Keyword(s):

Guinea Pig ◽

Signal To Noise Ratio ◽

Spotted Fever ◽

Clinical Signs ◽

Enzyme Linked Immunosorbent Assay ◽

Spotted Fever Group ◽

Rickettsia Parkeri ◽

Rickettsial Infections ◽

Spotted Fever Group Rickettsia ◽

Guinea Pig Model

Based on limited serological studies, at least 10% of the US population has been exposed to spotted fever group Rickettsia (SFGR) species. The immunofluorescence antibody assay (IFA) has been the gold standard for the serodiagnosis of rickettsial infections such as spotted fever rickettsiosis (SFR). However, the IFA is semi-quantitative and subjective, requiring a high level of expertise to interpret it correctly. Here, we developed an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Rickettsia parkeri infection in the guinea pig. Our ELISA is an objective, quantitative, and high-throughput assay that shows greater sensitivity and resolution in observed titers than the IFA. We methodically optimized relevant parameters in sequence for optimal signal-to-noise ratio and low coefficient of variation% values. We used a guinea pig model as it is a part of our overall research efforts to understand the immunological and clinical response to SFGR species after tick transmission. Guinea pigs are a useful model to study SFR and show clinical signs of SFR, such as fever and eschars. We anticipate that this assay will be easily adapted to other hosts, including humans and other SFGR species.

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“Rickettsia amblyommii” Induces Cross Protection Against Lethal Rocky Mountain Spotted Fever in a Guinea Pig Model

Vector-Borne and Zoonotic Diseases ◽

10.1089/vbz.2014.1575 ◽

2014 ◽

Vol 14 (8) ◽

pp. 557-562 ◽

Cited By ~ 19

Author(s):

Lucas S. Blanton ◽

Nicole L. Mendell ◽

David H. Walker ◽

Donald H. Bouyer

Keyword(s):

Guinea Pig ◽

Spotted Fever ◽

Rocky Mountain ◽

Cross Protection ◽

Pig Model ◽

Rocky Mountain Spotted Fever ◽

Rickettsia Amblyommii ◽

Guinea Pig Model

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Complementation ofRickettsia rickettsiiRelA/SpoT Restores a Nonlytic Plaque Phenotype

Infection and Immunity ◽

10.1128/iai.00048-11 ◽

2011 ◽

Vol 79 (4) ◽

pp. 1631-1637 ◽

Cited By ~ 20

Author(s):

Tina R. Clark ◽

Damon W. Ellison ◽

Betsy Kleba ◽

Ted Hackstadt

Keyword(s):

Active Site ◽

Stop Codon ◽

Spotted Fever ◽

Single Gene ◽

Premature Stop Codon ◽

Spotted Fever Group ◽

Single Mutation ◽

Spontaneous Mutant ◽

Spotted Fever Group Rickettsiae ◽

Plaque Phenotype

ABSTRACTSpotted fever group rickettsiae are known to produce distinct plaque phenotypes. Strains that cause lytic infections in cell culture form clear plaques, while nonlytic strains form opaque plaques in which the cells remain intact. Clear plaques have historically been associated with more-virulent species or strains of spotted fever group rickettsiae. We have selected spontaneous mutant pairs from two independent strains ofRickettsia rickettsii, the virulent R strain and the avirulent Iowa strain. A nonlytic variant ofR. rickettsiiR, which typically produces clear plaques, was isolated and stably maintained. A lytic variant of the Iowa strain, which characteristically produces opaque plaques, was also selected and maintained. Genomic resequencing of the variants identified only a single gene disrupted in each strain. In both cases, the mutation was in a gene annotated asrelA/spoT-like. In the Iowa strain, a single mutation introduced a premature stop codon upstream from region encoding the predicted active site of RelA/SpoT and caused the transition to a lytic plaque phenotype. InR. rickettsiiR, the nonlytic plaque phenotype resulted from a single-nucleotide substitution that shifted a tyrosine residue to histidine near the active site of the enzyme. The intactrelA/spoTgene thus occurred in variants with the nonlytic plaque phenotype. Complementation of the truncatedrelA/spoTgene in the Iowa lytic plaque variant restored the nonlytic phenotype. TherelA/spoTmutations did not affect the virulence of either strain in a Guinea pig model of infection; R strain lytic and nonlytic variants both induced fever equally, and the mutation in Iowa to a lytic phenotype did not cause them to become virulent.

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