The Role of luxS in Histophilus somni Virulence and Biofilm Formation.
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ABSTRACT:
SUBMITTER: Pan Y
PROVIDER: S-EPMC7822144 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.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.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.
| S-EPMC3224263 | biostudies-literature
Project description: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.
| S-EPMC2863524 | biostudies-literature
Project description:Histophilus somni is a Gram-negative bacterium that is associated with a disease complex (termed histophilosis) that can produce several clinical syndromes predominantly in cattle, but also in sheep. Histophilosis is well described in North America, Canada, and in some European countries. In Brazil, histophilosis has been described in cattle with respiratory, reproductive, and systemic disease, with only one case described in sheep. This report describes the occurrence of Histophilus somni-associated disease in sheep from Southern Brazil. Eight sheep with different clinical manifestations from five farms were investigated by a combination of pathological and molecular diagnostic methods to identify additional cases of histophilosis in sheep from Brazil. The principal pathological lesions were thrombotic meningoencephalitis, fibrinous bronchopneumonia, pulmonary abscesses, and necrotizing myocarditis. The main clinical syndromes associated with H. somni were thrombotic meningoencephalitis (n=4), septicemia (n=4), bronchopneumonia (n=4), and myocarditis (n=3). H. somni DNA was amplified from multiple tissues of all sheep with clinical syndromes of histophilosis; sequencing confirmed the PCR results. Further, PCR assays to detect Pasteurella multocida and Mannheimia haemolytica were negative. These findings confirmed the participation of H. somni in the clinical syndromes investigated during this study, and adds to the previous report of histophilosis in sheep from Brazil.
| S-EPMC6066786 | biostudies-literature
Project description:The sudden death of three calves, one diarrheic calf, and one aborted fetus from four farms in southern Brazil was investigated. Two Histophilus somni-associated syndromes were identified: systemic histophilosis (n = 4) and abortion (n = 1). The principal pathological findings included vasculitis, meningoencephalitis with thrombosis, necrotizing myocarditis, renal infarctions, hepatic abscesses, and bronchopneumonia. PCR assays were used to amplify specific amplicons of the ovine herpesvirus 2, bovine herpesvirus 1 and -5, Listeria monocytogenes, H. somni, and pestivirus; bovine group A rotavirus (BoRV-A) and bovine coronavirus (BCoV) were investigated in calves with diarrhea. H. somni DNA was amplified in tissues from all calves and the brain of the aborted fetus with pathological alterations consistent with histophilosis. All other PCR assays were negative; BoRV-A and BCoV were not identified. These findings confirm the participation of H. somni in the pathological alterations observed in this study and represent the first description of histophilosis in cattle from Brazil.
| S-EPMC7088796 | biostudies-literature
Project description:Capnocytophaga ochracea is present in the dental plaque biofilm of patients with periodontitis. Biofilm cells change their phenotype through quorum sensing in response to fluctuations in cell-population density. Quorum sensing is mediated by auto-inducers (AIs). AI-2 is involved in intercellular signaling, and production of its distant precursor is catalyzed by LuxS, an enzyme involved in the activated methyl cycle. Our aim was to clarify the role of LuxS in biofilm formation by C. ochracea. Two luxS-deficient mutants, TmAI2 and LKT7, were constructed from C. ochracea ATCC 27872 by homologous recombination. The mutants produced significantly less AI-2 than the wild type. The growth rates of these mutants were similar to that of the wild-type in both undiluted Tryptic soy broth and 0.5 × Tryptic soy broth. However, according to crystal violet staining, they produced significantly less biofilm than the wild type. Confocal laser scanning microscopy and scanning electron microscopy showed that the biofilm of the TmAI2 strain had a rougher structure than that of the wild type. Complementation of TmAI-2 with extrinsic AI-2 from the culture supernatant of wild-type strain did not restore biofilm formation by the TmAI2 strain, but complementation of LKT7 strain with luxS partially restored biofilm formation. These results indicate that LuxS is involved in biofilm formation by C. ochracea, and that the attenuation of biofilm formation by the mutants is likely caused by a defect in the activated methyl cycle rather than by a loss of AI-2.
| S-EPMC4723239 | biostudies-literature
Project description:Haemophilus parasuis (H. parasuis) is a kind of opportunistic pathogen of the upper respiratory tract of piglets. Under certain circumstances, virulent strains can breach the mucosal barrier and enter the bloodstream, causing severe Glässer's disease. Many virulence factors are found to be related to the pathogenicity of H. parasuis strain, but the pathogenic mechanism remains unclear. LuxS/AI-2, as a kind of very important quorum sensing system, affects the growth characteristics, biofilm formation, antibiotic production, virulence, and metabolism of different strains. In order to investigate the effect of luxS/AI-2 quorum sensing system on the virulence of H. parasuis, a deletion mutant strain (ΔluxS) and complemented strain (C-luxS) were constructed and characterized. The results showed that the luxS gene participated in regulating and controlling stress resistance, biofilm formation and virulence. Compared with wild-type strain, ΔluxS strain decreased the production of AI-2 molecules and the tolerance toward oxidative stress and heat shock, and it reduced the abilities of autoagglutination, hemagglutination, and adherence, whereas it increased the abilities to form biofilm in vitro. In vivo experiments showed that ΔluxS strain attenuated its virulence about 10-folds and significantly decreased its tissue burden of bacteria in mice, compared with the wild-type strain. Taken together, the luxS/AI-2 quorum sensing system in H. parasuis not only plays an important role in growth and biofilm formation, but also affects the pathogenicity of H. parasuis.
| S-EPMC6434701 | biostudies-literature
Project description:Histophilus somni (Haemophilus somnus) is an important pathogen of cattle that is responsible for respiratory disease, septicemia, and systemic diseases such as thrombotic meningoencephalitis, myocarditis, and abortion. A variety of virulence factors have been identified in H. somni, including compositional and antigenic variation of the lipooligosaccharide (LOS). Phosphorylcholine (ChoP) has been identified as one of the components of H. somni LOS that undergoes antigenic variation. In this study, five genes (lic1ABCD(Hs) and glpQ) with homology to genes responsible for ChoP expression in Haemophilus influenzae LOS were identified in the H. somni genome. An H. somni open reading frame (ORF) with homology to H. influenzae lic1A (lic1A(Hi)) contained a variable number of tandem repeats (VNTR). However, whereas the tetranucleotide repeat 5'-CAAT-3' is present in lic1A(Hi), the VNTR in H. somni lic1A (lic1A(Hs)) consisted of 5'-AACC-3'. Due to the propensity of VNTR to vary during replication and cause the ORF to shift in and out of frame with the upstream start codon, the VNTR were deleted from lic1A(Hs) to maintain the gene constitutively on. This construct was cloned into Escherichia coli, and functional enzyme assays confirmed that lic1A(Hs) encoded a choline kinase, and that the VNTR were not required for expression of a functional gene product. Variation in the number of VNTR in lic1A(Hs) correlated with antigenic variation of ChoP expression in H. somni strain 124P. However, antigenic variation of ChoP expression in strain 738 predominately occurred through variable extension/truncation of the LOS outer core. These results indicated that the lic1(Hs) genes controlled expression of ChoP on the LOS, but that in H. somni there are two potential mechanisms that account for antigenic variation of ChoP.
| S-EPMC3569003 | biostudies-literature
Project description:Histophilus somni is an opportunistic pathogen responsible for respiratory and systemic diseases of cattle and sheep. Rapid and accurate detection of H. somni is essential to distinguish H. somni from other potential pathogens for proper control and treatment of infections. Nanomaterial optical fiber biosensors (NOFS) recognize analyte interactions, such as DNA hybridization, with high specificity and sensitivity, and were applied to detect H. somni DNA in culture and clinical samples. An ionic self-assembled multilayer (ISAM) film was fabricated on a long-period grating optical fiber, and a biotinylated, nucleotide probe complementary to the H. somni 16S rDNA gene was coupled to the ISAM film. Exposure of the ISAM::probe to ⩾100 killed cells of H. somni strain 2336 without DNA amplification resulted in attenuation of light transmission of ⩾9.4%. Exposure of the complexed fiber to Escherichia coli or non- H. somni species of Pasteurellaceae reduced light transmission by ⩽3.4%. Exposure of the ISAM::probe to blood, bronchoalveolar fluid, or spleen from mice or calves infected with H. somni resulted in ⩾24.3% transmission attenuation. The assay correctly detected all 6 strains of H. somni tested from culture, or tissues from 3 separate mice and calves tested in duplicate. Six heterologous strains (representing 6 genera) reacted at below the cutoff value of 4.87% attenuation of light transmission. NOFS detected at least 100 H. somni cells without DNA amplification within 45 min with high specificity. Although different fibers could vary in signal sensitivity, this did not affect the sensitivity or specificity of the assay.
| S-EPMC6505835 | biostudies-literature
Project description:Autoinducer-2 (AI-2) molecules are one class of signalling molecules involved in gene regulation dependent on population density in a mechanism commonly referred to as quorum sensing (QS). AI-2 is produced by the methylthioadenosine/S-adenosyl-homocysteine nucleosidase LuxS. In the present study, we characterise the function of bifidobacterial LuxS proteins to address the question whether these economically important bacteria are able to perform QS communication. All publically available genome sequences of bifidobacteria harbour putative luxS genes. The deduced amino acid sequences are well conserved in the genus and show good homology to the LuxS protein of the prototypical AI-2 producer Vibrio harveyi. The luxS genes of three bifidobacterial strains were successfully expressed in AI-2-negative Escherichia coli DH5α. Supernatants of these recombinant E. coli strains contained significant AI-2 activity. In initial experiments, we failed to detect AI-2 activity in supernatants of bifidobacteria grown in MRSc. High concentration of glucose as well as acidic pH had strong inhibitory effects on AI-2 activity. AI-2 activity could be detected when lower volumes of supernatants were used in the assay. Homologous overexpression of luxS in Bifidobacterium longum NCC2705 increased AI-2 levels in the supernatant. Furthermore, over-expression of luxS or supplementation with AI-2-containing supernatants enhanced biofilm formation of B. longum NCC2705. Collectively, these results suggest that bifidobacteria indeed harbour functional luxS genes that are involved in the production of AI-2-like molecules. To the best of our knowledge, this represents the first report on AI-2 activity produced by bifidobacteria. Self-produced AI-2 activity as well as AI-2-like molecules of other bacteria of the intestinal tract may have a regulatory function in biofilm formation and host colonization by bifidobacteria.
| S-EPMC3914940 | biostudies-literature
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.
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