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Histophilus somni IbpA DR2/Fic in virulence and immunoprotection at the natural host alveolar epithelial barrier.

ABSTRACT: Newly recognized Fic family virulence proteins may be important in many bacterial pathogens. To relate cellular mechanisms to pathogenesis and immune protection, we studied the cytotoxicity of the Histophilus somni immunoglobulin-binding protein A (IbpA) direct repeat 2 Fic domain (DR2/Fic) for natural host target cells. Live virulent IbpA-producing H. somni strain 2336, a cell-free culture supernatant (CCS) of this strain, or recombinant DR2/Fic (rDR2/Fic) caused dramatic retraction and rounding of bovine alveolar type 2 (BAT2) epithelial cells. IbpA-deficient H. somni strain 129Pt and a Fic motif His(298)Ala mutant rDR2/Fic protein were not cytotoxic. The cellular mechanism of DR2/Fic cytotoxicity was demonstrated by incubation of BAT2 cell lysates with strain 2336 CCS or rDR2/Fic in the presence of [alpha-(32)P]ATP, which resulted in adenylylation of Rho GTPases and cytoskeletal disruption. Since IbpA is not secreted by type III or type IV secretion systems, we determined whether DR2/Fic entered the host cytoplasm to access its Rho GTPase targets. Although H. somni did not invade BAT2 cells, DR2/Fic was internalized by cells treated with H. somni, CCS, or the rDR2/Fic protein, as shown by confocal immunomicroscopy. Transwell bacterial migration assays showed that large numbers of strain 2336 bacteria migrated between retracted BAT2 cells, but IbpA-deficient strain 129Pt did not cross a monolayer unless the monolayer was pretreated with strain 2336 CCS or rDR2/Fic protein. Antibody to rDR2/Fic or passively protective convalescent-phase serum blocked IbpA-mediated cytotoxicity and inhibited H. somni transmigration across BAT2 monolayers, confirming the role of DR2/Fic in pathogenesis and corresponding to the results for in vivo protection in previous animal studies.

SUBMITTER: Zekarias B

PROVIDER: S-EPMC2863524 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Histophilus somni IbpA DR2/Fic in virulence and immunoprotection at the natural host alveolar epithelial barrier.

Zekarias Bereket B Mattoo Seema S Worby Carolyn C Lehmann Jason J Rosenbusch Ricardo F RF Corbeil Lynette B LB

Infection and immunity 20100222 5

Newly recognized Fic family virulence proteins may be important in many bacterial pathogens. To relate cellular mechanisms to pathogenesis and immune protection, we studied the cytotoxicity of the Histophilus somni immunoglobulin-binding protein A (IbpA) direct repeat 2 Fic domain (DR2/Fic) for natural host target cells. Live virulent IbpA-producing H. somni strain 2336, a cell-free culture supernatant (CCS) of this strain, or recombinant DR2/Fic (rDR2/Fic) caused dramatic retraction and roundin ...[more]

PMID: 20176790

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Project description:A new family of adenylyltransferases, defined by the presence of a Fic domain, was recently discovered to catalyze the addition of adenosine monophosphate (AMP) to Rho GTPases (Yarbrough, M. L., Li, Y., Kinch, L. N., Grishin, N. V., Ball, H. L., and Orth, K. (2009) Science 323, 269-272; Worby, C. A., Mattoo, S., Kruger, R. P., Corbeil, L. B., Koller, A., Mendez, J. C., Zekarias, B., Lazar, C., and Dixon, J. E. (2009) Mol. Cell 34, 93-103). This adenylylation event inactivates Rho GTPases by preventing them from binding to their downstream effectors. We reported that the Fic domain(s) of the immunoglobulin-binding protein A (IbpA) from the pathogenic bacterium Histophilus somni adenylylates mammalian Rho GTPases, RhoA, Rac1, and Cdc42, thereby inducing host cytoskeletal collapse, which allows H. somni to breach alveolar barriers and cause septicemia. The IbpA-mediated adenylylation occurs on a functionally critical tyrosine in the switch 1 region of these GTPases. Here, we conduct a detailed characterization of the IbpA Fic2 domain and compare its activity with other known Fic adenylyltransferases, VopS (Vibrio outer protein S) from the bacterial pathogen Vibrio parahaemolyticus and the human protein HYPE (huntingtin yeast interacting protein E; also called FicD). We also included the Fic domains of the secreted protein, PfhB2, from the opportunistic pathogen Pasteurella multocida, in our analysis. PfhB2 shares a common domain architecture with IbpA and contains two Fic domains. We demonstrate that the PfhB2 Fic domains also possess adenylyltransferase activity that targets the switch 1 tyrosine of Rho GTPases. Comparative kinetic and phylogenetic analyses of IbpA-Fic2 with the Fic domains of PfhB2, VopS, and HYPE reveal important aspects of their specificities for Rho GTPases and nucleotide usage and offer mechanistic insights for determining nucleotide and substrate specificities for these enzymes. Finally, we compare the evolutionary lineages of Fic proteins with those of other known adenylyltransferases.

Project description:Histophilus somni is a Gram-negative bacterial organism that acts as an opportunistic pathogen and is a fastidious member of the Pasteurellaceae family associated with diseases of respiratory, reproductive, cardiac, and other tissues of ruminants. We identified an intervening sequence (IVS) embedded in all five copies of the 23S rRNA gene in the closed genome sequence of the H. somni isolate USDA-ARS-USMARC-63250 that may play an important role in affecting the biology of the organism. Sequencing the RNA from this isolate shows that most of the IVS is cleaved from the transcript, resulting in independent fragments of this structural rRNA that remain functional within the bacterial ribosome. The IVS lies between positions 1170 and 1278 bp of the 3,017-bp gene and exhibits self-complementarity between its 5' and 3' ends that predicts a stem-loop structure interrupting helix-45 in the transcribed 23S rRNA. Excision removes a 94-nucleotide (nt) stem-loop structure that displays an unusual 1-nt 3' end overhang instead of the more typical 2-nt overhang commonly observed at the ends of other excised IVS stem-loops. A comparison with genomes of other H. somni isolates indicates that this IVS is highly conserved, with 31 of 32 complete genomes having similar interruptions of canonical 23S rRNA genes. The potential biological effects of either the released IVS or the fragmentation of the functional 23S rRNA are unknown, but fragmentation may enhance rRNA degradation in ways that contribute to the regulation of gene expression. IMPORTANCE The genome biology underlying H. somni virulence, pathogenicity, environmental adaptability, and broad tissue tropism is understood poorly. We identified a novel H. somni 109-nt IVS stem-loop structure, of which the central portion is excised from the 23S rRNA transcript, resulting in the fragmentation of this rRNA in the H. somni isolate USDA-ARS-USMARC-63250 and the release of a 94-nt structured RNA of unknown function. We determined that this peculiar rRNA biology is widespread among sequenced H. somni isolates, suggesting it has importance to organism biology. The fragmented 23S rRNA molecules remain functional in the ribosome, given that the isolate grows in culture. The structured excised portion of the IVS, presumably due to the action of the endoribonuclease III, has an unusual 1-nt 3' end overhang. This newly discovered H. somni 23S rRNA fragmentation may enhance rRNA degradation providing a previously unrecognized avenue for regulating H. somni biological processes.

Project description:Background and aimHistophilus somni is a Gram-negative bacterium belonging to the Pasteurellaceae family that can cause bovine histophilosis. Histophilus may act as a commensal or opportunistic bacterial cattle pathogen. Comparing genomes of the pathogenic strain 2336 with the non-pathogenic preputial 129Pt isolate revealed some putative virulence factors. The study of the complete genomes of H. somni strains circulating in Russia has never been conducted before. This study aimed to identify genetic features of the H. somni strains isolated in Russia and evaluate the possibility of using strains for vaccine development.Materials and methodsThree strains of H. somni were isolated from different sources. Strain 188-VIEV was isolated from a vaginal swab sample of cattle with endometritis. 532-VIEV and 551-VIEV were cultured from the cryopreserved bull semen samples imported from Canada. Histophilus somni strain ATCC 700025 provided by ATCC (American Type Culture Collection) was also used in the study. DNA extraction was performed using QIAamp DNA Mini Kit (QIAGEN, USA). The whole-genome sequencing of the four strains was performed using Illumina Miseq. The comparison of the resulting sequences with the complete genomes of H. somni 2336 and 129Pt, and detection of the resistance genes and virulence factors, was performed using the ResFinder and Virulence Factor Database web services.ResultsThe genome size of the samples varied from 1.9 to 2.3 Mb. The number of coding sequences varied from 1795 to 2256. The average sequence density was 90%. The total guanine-cytosine (GC) content was 36.8%-37.2%, which coincided with data previously obtained for H. somni. Three out of four studied strains encoded putative virulence factors such as filamentous hemagglutinin homologs, lipooligosaccharide biosynthesis proteins, and proteins involved in iron transport and utilization. The Ser83Ile substitution was identified in the DNA topoisomerase II (gyrA) in H. somni strains 532-VIEV and 551-VIEV cultured from bull semen which led to resistance to fluoroquinolones. The gene (AAC-6-Ia + APH-2'') encoding a bifunctional aminoglycoside modification enzyme was detected in strain 551-VIEV.ConclusionStrains with virulence genes identified could be candidates for designing vaccines and potentially represent antigen sources. The results show that antibiotic-resistant H. somni can be spread with semen used for artificial insemination.

Dataset Information

Histophilus somni IbpA DR2/Fic in virulence and immunoprotection at the natural host alveolar epithelial barrier.

Publications

Histophilus somni IbpA DR2/Fic in virulence and immunoprotection at the natural host alveolar epithelial barrier.

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