Correction: Influence of the shared epitope on the elicitation of experimental autoimmune arthritis biomarkers.

[This corrects the article DOI: 10.1371/journal.pone.0250177.].

The role of uspE in virulence and biofilm formation by Histophilus somni.

UspE is a global regulator in Escherichia coli. To study the function of Histophilus somni uspE, strain 2336::TnuspE was identified from a bank of mutants generated with EZ::Tn5™ Tnp Transposome™ that were biofilm deficient. The 2336::TnuspE mutant was highly attenuated in mice, the electrophoretic profile of its lipooligosaccharide (LOS) indicated the LOS was truncated, and the mutant was significantly more serum-sensitive compared to the wildtype strain. In addition to forming a deficient biofilm, exopolysaccharide (EPS) production was also compromised, but the electrophoretic profile of outer membrane proteins was not altered. RNA sequence analysis revealed that the transcription levels of some stress response chaperones, transport proteins, and a large number of ribosomal protein genes in 2336::TnuspE were significantly differentially regulated compared to strain 2336. Therefore, uspE may differentially function in direct and indirect expression of H. somni genes, but its attenuation may be linked to poor biofilm formation and rapid clearance of the bacteria resulting from a compromised LOS structure. Our results support that uspE is a global stress regulatory gene in H. somni.

Influence of the shared epitope on the elicitation of experimental autoimmune arthritis biomarkers.

Our previous studies have shown that inoculation of the oral cavity of "humanized" B6.DR1/4 mice with the periodontal pathogen Porphyromonas gingivalis results in an increase in the percentage of circulating Th17 cells, loss of bone and an exacerbation of experimental autoimmune arthritis. The aim of this study was to assess the role played by the human HLA-DRß molecule containing the shared epitope supplied as a transgene to I-A˚ (murine class II null) C57BL/6 (B6) mice in driving these findings. We compared various immune response parameters as well as alveolar and peri-articular bone loss between humanized B6.DR1 (or B6.DR4) mice and their WT (B6) counterparts. We found that the presence of the shared epitope in the context of inoculation with P. gingivalis enhanced the percentage of Th17 cells generated, dramatically enhanced bone loss and importantly allowed for the generation of CCP2⁺ ACPAs that are not found in C57BL/6 or DBA/1 arthritic mouse serum. Due to the exceedingly complex nature of environmental factors impacting on genetic elements, it has been difficult to unravel mechanisms that drive autoimmune arthritis in susceptible individuals. The findings in this study may provide one small piece of this puzzle that can help us to better understand part of this complexity.

The Role of luxS in Histophilus somni Virulence and Biofilm Formation.

S-Ribosylhomocysteinase (LuxS) is required for the synthesis of the autoinducer-2 (AI-2) quorum-sensing signaling molecule in many Gram-negative bacteria. The bovine (and ovine) opportunistic pathogen Histophilus somni contains luxS and forms a biofilm containing an exopolysaccharide (EPS) in the matrix. Since biofilm formation is regulated by quorum sensing in many bacteria, the roles of luxS in H. somni virulence and biofilm formation were investigated. Although culture supernatants from H. somni were ineffective at inducing bioluminescence in the Vibrio harveyi reporter strain BB170, H. somniluxS complemented the biosynthesis of AI-2 in the luxS-deficient Escherichia coli strain DH5α. H. somni strain 2336 luxS was inactivated by transposon mutagenesis. RNA expression profiles revealed that many genes were significantly differentially expressed in the luxS mutant compared to that in the wild-type, whether the bacteria were grown planktonically or in a biofilm. Furthermore, the luxS mutant had a truncated and asialylated lipooligosaccharide (LOS) and was substantially more serum sensitive than the wild-type. Not surprisingly, the luxS mutant was attenuated in a mouse model for H. somni virulence, and some of the altered phenotypes were partially restored after the mutation was complemented with a functional luxS However, no major differences were observed between the wild-type and the luxS mutant in regard to outer membrane protein profiles, biofilm formation, EPS production, or intracellular survival. These results indicate that luxS plays a role in H. somni virulence in the context of LOS biosynthesis but not biofilm formation or other phenotypic properties examined.

Histophilus somni Survives in Bovine Macrophages by Interfering with Phagosome-Lysosome Fusion but Requires IbpA for Optimal Serum Resistance.

Histophilus somni is capable of intracellular survival within professional phagocytic cells, but the mechanism of survival is not understood. The Fic motif within the direct repeat (DR1)/DR2 domains of the IbpA fibrillary network protein of H. somni is cytotoxic to epithelial and phagocytic cells, which may interfere with the bactericidal activity of these cells. To determine the contribution of IbpA and Fic to resistance to host defenses, H. somni strains and mutants that lacked all or a region of ibpA (including the DR1/DR2 regions) were tested for survival in bovine monocytic cells and for serum susceptibility. An H. somni mutant lacking IbpA, but not the DR1/DR2 region within ibpA, was more susceptible to killing by antiserum than the parent, indicating that the entire protein was associated with serum resistance. H. somni strains expressing IbpA replicated in bovine monocytes for at least 72 h and were toxic for these cells. Virulent strain 2336 mutants lacking the entire ibpA gene or both DR1 and DR2 were not toxic to the monocytes but still survived within the monocytes for at least 72 h. Monitoring of intracellular trafficking of H. somni with monoclonal antibodies to phagosomal markers indicated that the early phagosomal marker early endosome antigen 1 colocalized with all isolates tested, but only strains that could survive intracellularly did not colocalize with the late lysosomal marker lysosome-associated membrane protein 2 and prevented the acidification of phagosomes. These results indicated that virulent isolates of H. somni were capable of surviving within phagocytic cells through interference in phagosome-lysosome maturation. Therefore, H. somni may be considered a permissive intracellular pathogen.

Bone loss and aggravated autoimmune arthritis in HLA-DRß1-bearing humanized mice following oral challenge with Porphyromonas gingivalis.

BACKGROUND: The linkage between periodontal disease and rheumatoid arthritis is well established. Commonalities among the two are that both are chronic inflammatory diseases characterized by bone loss, an association with the shared epitope susceptibility allele, and anti-citrullinated protein antibodies. METHODS: To explore immune mechanisms that may connect the two seemingly disparate disorders, we measured host immune responses including T-cell phenotype and anti-citrullinated protein antibody production in human leukocyte antigen (HLA)-DR1 humanized C57BL/6 mice following exposure to the Gram-negative anaerobic periodontal disease pathogen Porphyromonas gingivalis. We measured autoimmune arthritis disease expression in mice exposed to P. gingivalis, and also in arthritis-resistant mice by flow cytometry and multiplex cytokine-linked and enzyme-linked immunosorbent assays. We also measured femoral bone density by microcomputed tomography and systemic cytokine production. RESULTS: Exposure of the gingiva of DR1 mice to P. gingivalis results in a transient increase in the percentage of Th17 cells, both in peripheral blood and cervical lymph nodes, a burst of systemic cytokine activity, a loss in femoral bone density, and the generation of anti-citrullinated protein antibodies. Importantly, these antibodies are not produced in response to P. gingivalis treatment of wild-type C57BL/6 mice, and P. gingivalis exposure triggered expression of arthritis in arthritis-resistant mice. CONCLUSIONS: Exposure of gingival tissues to P. gingivalis has systemic effects that can result in disease pathology in tissues that are spatially removed from the initial site of infection, providing evidence for systemic effects of this periodontal pathogen. The elicitation of anti-citrullinated protein antibodies in an HLA-DR1-restricted fashion by mice exposed to P. gingivalis provides support for the role of the shared epitope in both periodontal disease and rheumatoid arthritis. The ability of P. gingivalis to induce disease expression in arthritis-resistant mice provides support for the idea that periodontal infection may be able to trigger autoimmunity if other disease-eliciting factors are already present.

Natural competence in Histophilus somni strain 2336.

Histophilus somni is an etiologic agent of shipping fever pneumonia, myocarditis, and other systemic diseases of bovines. Virulence factors that have been identified in H. somni include biofilm formation, lipooligosaccharide phase variation, immunoglobulin binding proteins, survival in phagocytic cells, and many others. However, to identify the genes responsible for virulence, an efficient mutagenesis system is needed. Mutagenesis of H. somni using allelic exchange is difficult, likely due to its tight restriction modification system. Mutagenesis by natural transformation in Haemophilus influenzae is well established and shows a strong bias for fragments containing specific uptake signal sequences (USS) within the genome. We hypothesized that natural transformation may also be possible in H. somni strain 2336 because its genome is over-represented with H. influenzae USS (5'-AAGTGCGGT-3') and contains most of the genes necessary for competence. H. somni strain 2336 was successfully transformed and mutated with genomic linear DNA from an H. somni mutant (738Δlob2a), which contains a kanamycin-resistance (Kan(R)) gene and the USS within lob2A. Although most of the competence genes found in H. influenzae were present in H. somni, comD and the 5' portion of comE were absent, which may account for the low transformation efficiency. The transformation efficiency of strain 2336 was greatest during mid-log growth phase and when cyclic adenosine monophosphate was added to the transformation medium. However, mutants were not isolated when strain 2336 was transformed with genomic DNA containing the same Kan(R) gene from H. somni luxS or uspE mutants, which lack the USS in these specific genes. Shuttle vector pNS3K was also naturally transformed into strain 2336, though at a lower efficiency. However, natural transformation with either H. somni linear DNA (2336Δlob2A) or pNS3K was unsuccessful with H. somni commensal strain 129Pt and several other disease isolates.

Identification, structure, and characterization of an exopolysaccharide produced by Histophilus somni during biofilm formation.

BACKGROUND: Histophilus somni, a gram-negative coccobacillus, is an obligate inhabitant of bovine and ovine mucosal surfaces, and an opportunistic pathogen responsible for respiratory disease and other systemic infections in cattle and sheep. Capsules are important virulence factors for many pathogenic bacteria, but a capsule has not been identified on H. somni. However, H. somni does form a biofilm in vitro and in vivo, and the biofilm matrix of most bacteria consists of a polysaccharide. RESULTS: Following incubation of H. somni under growth-restricting stress conditions, such as during anaerobiosis, stationary phase, or in hypertonic salt, a polysaccharide could be isolated from washed cells or culture supernatant. The polysaccharide was present in large amounts in broth culture sediment after H. somni was grown under low oxygen tension for 4-5 days (conditions favorable to biofilm formation), but not from planktonic cells during log phase growth. Immuno-transmission electron microscopy showed that the polysaccharide was not closely associated with the cell surface, and was of heterogeneous high molecular size by gel electrophoresis, indicating it was an exopolysaccharide (EPS). The EPS was a branched mannose polymer containing some galactose, as determined by structural analysis. The mannose-specific Moringa M lectin and antibodies to the EPS bound to the biofilm matrix, demonstrating that the EPS was a component of the biofilm. The addition of N-acetylneuraminic acid to the growth medium resulted in sialylation of the EPS, and increased biofilm formation. Real-time quantitative reverse transcription-polymerase chain reaction analyses indicated that genes previously identified in a putative polysaccharide locus were upregulated when the bacteria were grown under conditions favorable to a biofilm, compared to planktonic cells. CONCLUSIONS: H. somni is capable of producing a branching, mannose-galactose EPS polymer under growth conditions favorable to the biofilm phase of growth, and the EPS is a component of the biofilm matrix. The EPS can be sialylated in strains with sialyltransferase activity, resulting in enhanced density of the biofilm, and suggesting that EPS and biofilm formation may be important to persistence in the bovine host. The EPS may be critical to virulence if the biofilm state is required for H. somni to persist in systemic sites.

Chemical modification of L-glutamine to alpha-amino glutarimide on autoclaving facilitates Agrobacterium infection of host and non-host plants: A new use of a known compound.

BACKGROUND: Accidental autoclaving of L-glutamine was found to facilitate the Agrobacterium infection of a non host plant like tea in an earlier study. In the present communication, we elucidate the structural changes in L-glutamine due to autoclaving and also confirm the role of heat transformed L-glutamine in Agrobacterium mediated genetic transformation of host/non host plants. RESULTS: When autoclaved at 121°C and 15 psi for 20 or 40 min, L-glutamine was structurally modified into 5-oxo proline and 3-amino glutarimide (α-amino glutarimide), respectively. Of the two autoclaved products, only α-amino glutarimide facilitated Agrobacterium infection of a number of resistant to susceptible plants. However, the compound did not have any vir gene inducing property. CONCLUSIONS: We report a one pot autoclave process for the synthesis of 5-oxo proline and α-amino glutarimide from L-glutamine. Xenobiotic detoxifying property of α-amino glutarimide is also proposed.

A genomic window into the virulence of Histophilus somni.

Histophilus somni is an obligate inhabitant of the respiratory and genital mucosal surfaces of bovines and ovines. An individual strain can be a primary pathogen, an opportunistic pathogen, or a commensal, but can also move between these classifications if introduced into an appropriate site (e.g. the lungs) under conditions that favor bacterial persistence. H. somni is one of the bacterial agents responsible for bovine respiratory disease complex and can also cause a variety of systemic diseases in cattle and sheep. Isolates from disease sites, such as the lungs, heart, and brain, express a wide array of virulence factors (including biofilm formation) designed to evade host defense mechanisms. By contrast, some isolates from the healthy genital tract often lack many of these virulence factors. The genomic sequences of two bovine isolates, one from pneumonic lung and the other from healthy prepuce, have aided in deciphering the differences in phenotype and virulence between the two strains, and reveal their striking genetic similarity to Haemophilus influenzae and other members of the Pasteurellaceae.

Histophilus somni biofilm formation in cardiopulmonary tissue of the bovine host following respiratory challenge.

Biofilms form in a variety of host sites following infection with many bacterial species. However, the study of biofilms in a host is hindered due to the lack of protocols for the proper experimental investigation of biofilms in vivo. Histophilus somni is an agent of respiratory and systemic diseases in bovines, and readily forms biofilms in vitro. In the present study the capability of H. somni to form biofilms in cardiopulmonary tissue following experimental respiratory infection in the bovine host was examined by light microscopy, transmission electron microscopy, immunoelectron microscopy of ultrathin cryosections, scanning electron microscopy of freeze-fractured samples, and fluorescent in situ hybridization. Biofilms were evident and most prominent in the myocardium, and were associated with a large amount of amorphous extracellular material. Furthermore, Pasteurella multocida was often cultured with H. somni from heart and lung samples. Transposon mutagenesis of H. somni strain 2336 resulted in the generation of mutants that expressed more or less biofilm than the parent strain. Six mutants deficient in biofilm formation had an insertion in the gene encoding for a homolog of filamentous haemagglutinin (FHA), predicted to be involved in attachment. Thus, this investigation demonstrated that H. somni is capable of forming a biofilm in its natural host, that such a biofilm may be capable of harboring other bovine respiratory disease pathogens, and that the genes responsible for biofilm formation can be identified by transposon mutagenesis.

Construction of a high-efficiency shuttle vector for Histophilus somni.

The genetic manipulation of Histophilus somni is limited due to its high-fidelity restriction-modification system. The broad host-range shuttle plasmid pLS88 is capable of transforming some strains of H. somni, but is an inefficient vector. We have constructed an improved version of pLS88, pNS3K, that transforms H. somni strain 2336 100-fold more efficiently than its predecessor. The transformation efficiency was further increased when pNS3K was isolated from H. somni and retransformed into the same strain. As proof of principle, the lipooligosaccharide biosynthesis gene lob-2A was cloned into pNS3K and expressed in H. somni strain 129Pt, which lacks this gene. Thus, pNS3K is a useful shuttle vector for H. somni and a potential vector for genetic manipulation of this bacterium.

Characterization and comparison of biofilm development by pathogenic and commensal isolates of Histophilus somni.

Histophilus somni (Haemophilus somnus) is an obligate inhabitant of the mucosal surfaces of bovines and sheep and an opportunistic pathogen responsible for respiratory disease, meningoencephalitis, myocarditis, arthritis, and other systemic infections. The identification of an exopolysaccharide produced by H. somni prompted us to evaluate whether the bacterium was capable of forming a biofilm. After growth in polyvinyl chloride wells a biofilm was formed by all strains examined, although most isolates from systemic sites produced more biofilm than commensal isolates from the prepuce. Biofilms of pneumonia isolate strain 2336 and commensal isolate strain 129Pt were grown in flow cells, followed by analysis by confocal laser scanning microscopy and scanning electron microscopy. Both strains formed biofilms that went through stages of attachment, growth, maturation, and detachment. However, strain 2336 produced a mature biofilm that consisted of thick, homogenous mound-shaped microcolonies encased in an amorphous extracellular matrix with profound water channels. In contrast, strain 129Pt formed a biofilm of cell clusters that were tower-shaped or distinct filamentous structures intertwined with each other by strands of extracellular matrix. The biofilm of strain 2336 had a mass and thickness that was 5- to 10-fold greater than that of strain 129Pt and covered 75 to 82% of the surface area, whereas the biofilm of strain 129Pt covered 35 to 40% of the surface area. Since H. somni is an obligate inhabitant of the bovine and ovine host, the formation of a biofilm may be crucial to its persistence in vivo, and our in vitro evidence suggests that formation of a more robust biofilm may provide a selective advantage for strains that cause systemic disease.

Agrobacterium-mediated genetic transformation of tea leaf explants: effects of counteracting bactericidity of leaf polyphenols without loss of bacterial virulence.

Tea is one of the major crops in Asia and Africa, and its improvement by genetic modification is important for economy of many tea-producing regions. Although somatic embryos derived from cotyledon explants have been transformed with Agrobacterium, the leaves of several commercially important tea cultivars have remained recalcitrant to transformation, largely due to bactericidal effect of polyphenols that are exuded by tea leaves in vitro. Moreover, the commonly used polyphenol adsorbents and antioxidants cannot overcome this problem. Leaf explants, however, are more desirable than cotyledon-derived somatic embryos, especially when it is necessary to further improve a selected elite and also retain its superior traits. Thus, we developed a procedure for Agrobacterium-mediated genetic transformation of tea leaf explants which is based on the presence of L-glutamine in the co-cultivation medium. We then showed that the transformation process is facilitated via a protective action of L-glutamine against bactericidal effects of leaf polyphenols without affecting the bacterial virulence (vir) gene expression.