Progress 09/01/07 to 08/31/11
Outputs
OUTPUTS: Histophilus somni (H. somni) is an etiologic agent of bronchopneumonia and systemic disease in bovines, and can form biofilms in vitro and in vivo. Thus, formation of a biofilm may be crucial to the persistence of H. somni in vivo. A bank of H. somni mutants were generated using the EZ::Tn5(KAN-2)Tnp Transposome (Epicentre), and screened for altered biofilm formation by crystal violet assay and scanning electron microscopy. The transposon insertion sites of mutants were identified by sequencing of inverse PCR products. Biofilm-deficient isolates with mutations in fhaB (filamentous hemagglutinin protein, fhaC (outer membrane transporter protein for secretion of the filamentous hemagglutinin), uspE (a universal stress protein essential for cell motility and aggregation), and tolC (type I secretion outer membrane protein) were obtained. A random screening of mutants with normal biofilm formation identified an isolate with a mutation in luxS, which is responsible for production of the autoinducer (AI) of the AI-2 quorum sensing pathway. Mutants were tested for virulence in a mouse model of septicemia and systemic disease. In this model, the uspE and luxS mutants were highly attenuated, causing no disease or clinical symptoms at a challenge dose of greater than 2.5 X 108 colony forming units [CFU]) (LD50 of the parent is less than 5 x 107 CFU). Cattle were immunized twice with each mutant and then challenged in the respiratory tract with 1010 CFU of a virulent strain. Controls were immunized with a commercial bacterin. Although some pathology still occurred in the lungs of immunized cattle (possibly due to the high challenge dose), both mutants provided substantially greater protection to the cattle than the commercial vaccine. The luxS and uspE mutants were both altered in lipooligosaccharide electrophoretic profile and exopolysaccharide production. H. somni luxS did not function in a Vibrio harveyi quorum sensing assay, although it did complement an E. coli luxS mutant indicating the H. somni LuxS was functional. The luxS mutant was also more serum susceptible than the parent. H. somni and Pasteurella multocida formed an excellent biofilm together when grown in a bioreactor, but Mannheimia haemolytica did not. However, microarray experiments showed that 230 genes from H. somni and 374 genes from M. haemolytica were differentially expressed when the bacteria were grown together planktonically; 40 of these genes were homologous to both species. Infection of the bovine host with H. somni resulted in differential expression of genes from the heart or lung, particularly genes associated with metabolic pathways associated with the immune response, inhibition of pathways associated with cell differentiation, hematological system development and function, growth factor activity, lipid metabolism, peroxisome proliferator‐activated receptor (PPAR) signaling, and activation of classical and alternative complement system pathways. To enhance site-specific mutagenesis in H. somni a natural transformation system was developed in which a mutation in chromosomal DNA from a mutant strain can be transferred to the same DNA site in the wildtype strain. PARTICIPANTS: PI-Thomas J. Inzana, is responsible for the design and execution of experiments. Dr. Inzana is responsible for reports and for communication on all manuscripts. Co-PI - Indra Sandal, is responsible for the design and execution of the experiments, and immediate supervision of graduate students working on the project. Dr. Sandal is responsible for preparing reports and manuscripts. Professional development: Co-PI Indra Sandal was Visiting Research Scientist (06/2010- 08/2010) at The Center for Biofilm Engineering, Montana State University, Bozeman, to work on H. somni biofilm formation in a drip flow reactor (DFR) with Dr. Darla Goeres. Dr. Sandal is now a research Assistant Professor at the University of Utah School of Medicine. Dr. Kent Scarratt, a large animal clinician, assisted in any bovine work that was done. Dr. Ed Swords assisted in studies and techniques concerning biofilm formation. Graduate Students: During this reporting period Nehal Shah was supported on this project to work on optimization of a mutagenesis system in H. somni by natural transformation and transposon mutagenesis. Mr. Shah also completed an NIH summer internship this past summer. Graduate student Yu Pan (not paid on the grant), and several undergraduate students (not paid on the grant) received training in transposon mutagenesis, sequencing, and biofilm formation from assisting on this project. TARGET AUDIENCES: Animal health pharmaceutical companies, bovine farmers, bovine veterinarians, microbiologists, and infectious disease researchers could all benefit from the information generated from this project. The information generated from this research would be of benefit in the classroom, particularly at the graduate level and senior undergraduate level. The information would also benefit those interested in biofilm research, Histophilus somni, and vaccine development. PROJECT MODIFICATIONS: Graduate student Nehal Shah has been recruited after a previous graduate student on the project suddenly left the program. The availability of our bovine practitioner to work with us on this project and to get cattle for the research was delayed due to scheduling conflicts and availability of animals free of antibodies to H. somni. Investigation was initiated into polymicrobial biofilms, which was productive in that it was determined that H. somni and P. multocida form an excellent biofilm together and were often co-isolated from the respiratory tract following challenge only with H. somni. Microarrays to examine gene expression by H. somni and M. haemolytica were initiated and provided evidence that further investigation into gene expression during polymicrobial infection is warranted.
Impacts
The results of this work indicate that the formation of a biofilm by H. somni is an important virulence factor and may be a requirement for this organism to cause disease. Multiple genes have been identified that contribute to biofilm formation, some of which are absent in a commensal strain unable to cause disease. An improved method for mutagenesis has been identified and applied to isolating mutants that are deficient in biofilm formation. Results from analysis of luxS and uspE mutants suggest these genes have regulatory functions that may control a wide variety of virulence factors. Further investigation into the properties of these mutants is warranted because both mutants were attenuated in cattle and could provide protection greater than that provided by a commercial bacterin. The efficiency in which H. somni and P. multocida form a biofilm together indicate these pathogens may form an efficient polymicrobial biofilm in the host. This polymicrobial interaction needs to be explored further to determine if this interaction contributes to resistance to antibiotic treatment and vaccination. The results of this work will be important for the development of new, more effective vaccines and diagnostic tests for bovine respiratory disease, and in determining the importance and role of a biofilm in disease due to H. somni. Transposon mutagenesis with EZ::Tn5Tnp Transposome is an efficient method for generating mutants of H. somni for identification of virulence factors and their role in bovine respiratory disease. However, natural transformation may further enhance site-specific mutagenesis of desired genes.
Publications
- Howard, M.D., L. Willis, W. Wakarchuk, F. St. Michael, A. Cox, W.T. Horne, R. Hontecilas, J. Bassaganya-Riera, E. Lorenz, and T.J. Inzana. 2011. Genetics and molecular specificity of sialylation of Histophilus somni lipooligosaccharide (LOS) and the effect of LOS sialylation on Toll-like receptor-4 signaling. Vet. Microbiol. 153:163-172.
- Sandal, I., T.J. Inzana, A. Molinaro, C. De Castro, J.Q. Shao, M.A. Apicella, A.D. Cox, F. St. Michael, and G. Berg. 2011. Identification, structure, and characterization of an exopolysaccharide produced by Histophilus somni during biofilm formation. BMC Microbiol. 11:186.
- Siddaramappa, S., J.F. Challacombe, A.J. Duncan, A.F. Gillaspy, M. Carson, J. Gipson, M. Gipson, J. Orvis, J. Zaitshik, G. Barnes, T.S. Brettin, D. Bruce, O. Chertkov, J.C. Detter, C.S. Han, R. Tapia, L.S. Thompson, D.W. Dyer, and T.J. Inzana. 2011. Genome sequence of Histophilus somni strain 2336 from bovine pneumonia and comparison to commensal strain 129Pt reveals extensive horizontal gene transfer and evolution of pathogenesis. BMC Genomics, In press.
- Inzana, T.J., I. Sandal, A. Molinaro, C. De Castro, Y. Pan, and S. Siddaramappa. 2011. Histophilus somni genetic factors and co-culture with other respiratory pathogens contribute to biofilm formation and virulence in the bovine host. Abst. P58. International Pasteurellaceae Conference 2011. Elsinore, Denmark.
- Shah, N., I. sandal, and T.J. Inzana. 2011. Novel methods of mutagenesis for Histophilus somni. American Society for Microbiology Virginia Branch 2011 Annual Meeting. Blacksburg, VA.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Histophilus somni (H. somni) is an etiologic agent of bronchopneumonia and systemic disease in bovines, and can form biofilms in vitro and in vivo. Thus, formation of a biofilm may be crucial to the persistence of H. somni in vivo. A bank of H. somni mutants was generated using the EZ::Tn5Tnp Transposome (Epicentre), and screened for altered biofilm formation by crystal violet assay and scanning electron microscopy. The transposon (Tn) insertion sites of these biofilm-defective mutants were identified by sequencing of inverse PCR products. A Swiss Webster mouse model of septicemia and systemic disease was used to assess the virulence of Tn mutants. Cattle are being inoculated to determine if the luxS and/or uspE mutants are capable of inducing protection against challenge with virulent strains. The information generated from this work has been presented at meetings, such as the American Society for Microbiology General Meeting, the Conference for Research Workers in Animal Diseases, The Prato Conference on Bacterial Diseases of Animals, and Annual Conference of the American Association of Bovine Practitioners. The results have also been presented in classes to Veterinary students at the Virginia-Maryland Regional College of Veterinary Medicine. PARTICIPANTS: PI-Thomas J. Inzana, is responsible for the design and execution of experiments. Dr. Inzana is responsible for reports and for communication on all manuscripts. Co-PI - Indra Sandal, is responsible for the design and execution of the experiments, and immediate supervision of graduate students working on the project. Dr. Sandal is responsible for preparing reports and manuscripts. Professional development: Co-PI Indra Sandal was Visiting Research Scientist (06/2010- 08/2010) at The Center for Biofilm Engineering, Montana State University, Bozeman, to work on H. somni biofilm formation in a drip flow reactor (DFR) with Dr. Darla Goeres. Dr. Goeres (Goeres et al., 2009 in Nature protocol) reported the use of a DFR to grow Pseudomonas aeruginosa as a biofilm close to the air-liquid interface to mimic the environment of lungs with cystic fibrosis. Graduate Student - During this reporting period Nehal Shah was supported on this project to work on optimization of a mutagenesis system in H. somni by natural transformation. Graduate student Yu Pan (not paid on the grant), and several undergraduate students (not paid on the grant) received training in transposon mutagenesis, sequencing, and biofilm formation from assisting on this project. TARGET AUDIENCES: Animal health pharmaceutical companies, bovine farmers, bovine veterinarians, microbiologists, and infectious disease researchers could all benefit from the information generated from this project. The information generated from this research would be of benefit in the classroom, particularly at the graduate level and senior undergraduate level. The information would also benefit those interested in biofilm research, Histophilus somni, and vaccine development. PROJECT MODIFICATIONS: Graduate student Nehal Shah has been recruited after a previous graduate student on the project suddenly left the program. The availability of our bovine practitioner to work with us on this project and to get cattle for the research has been delayed due to scheduling conflicts and availability of animals free of antibodies to H. somni. We are collaborating with Boehringer-Ingelheim Vetmedica, Inc. (BIVI) to determine if the luxS and/or uspE mutants are capable of inducing protection against challenge with virulent strains in cattle.
Impacts
Screening of the H. somni mutant bank for isolates altered in biofilm formation resulted in the identification of biofilm-deficient isolates with mutations in fhaC (outer membrane transporter protein for secretion of the filamentous hemagglutinin), uspE (a universal stress protein essential for cell motility and aggregation), and tolC (encoding the type I secretion outer membrane protein). Furthermore, a random screening of mutants with normal biofilm formation identified an isolate with a mutation in luxS, which is responsible for production of the autoinducer (AI) of the AI-2 quorum sensing pathway that is present in most Gram-negative bacteria. luxS and quorum sensing have been shown to be important in biofilm formation in some bacteria, such as Pseudomonas aeruginosa (P. aeruginosa), and virulence in others, such as Haemophilus influenzae and Vibrio cholerae. The uspE and luxS mutants were highly attenuated in mice, causing no disease or clinical symptoms at a challenge dose of >2.5 X 108 colony forming units [CFU]) (LD50 of the parent is <5 x 107 CFU). Of interest though, was following the first immunization with the luxS mutant, most mice (9 of 15) went into shock and died on the same day following a second immunization with the mutant. Transposon mutagenesis with EZ::Tn5Tnp Transposome is an efficient method for generating mutants of H. somni to identify virulence factors and their role in bovine respiratory disease. The results of this work indicate that the formation of a biofilm by H. somni is an important virulence factor and may be required by this organism to cause disease. Multiple genes have been identified that contribute to biofilm formation, some of which are absent in a commensal strain unable to cause disease. An improved method for mutagenesis has been identified and applied to isolating mutants that are deficient in biofilm formation. The results of this work will be important for the development of new, more effective vaccines and diagnostic tests for this pathogen, and in determining the importance and role of a biofilm in disease due to H. somni.
Publications
- Sandal I, and Inzana TJ. Identification of genes involved in biofilm formation and virulence in Histophilus somni. CRWAD Meeting, Chicago, IL. Abstract 124. Dec. 6 to 8, 2009.
- Sandal I, El-hadidy M, and Inzana TJ. Identification of genes involved in Histophilus somni biofilm formation by transposon mutagenesis. 5th ASM Conference on Biofilms, Cancun, Mexico. Nov. 15 to 19, 2009.
- Sandal I, El-Hadidy M, and Inzana TJ. Preliminary characterization of Histophilus somni mutants deficient in biofilm formation or attenuated in virulence 43rd Annual Conference of the American Association of Bovine Practitioners and 2010 Academy of Veterinary Consultants summer meeting, Albuquerque, New Mexico. Aug. 15 to 18, 2010.
- Inzana TJ, Sandal I, Elswaifi SF, Scarratt K, Howard M, Willis L, Wakarchuk W, St. Michael F, Cox A, Balyan R, and Lorenz E. The Contribution of Histophilus somni (Haemophilus somnus) lipooligosaccharide to bacterial virulence and host interactions. Prato Conference on the Pathogenesis of Bacterial Diseases of Animals. Abstract 13. Prato, Italy Oct. 6 to 9, 2010.
- Sandal I, Inzana TJ. A genomic window into the virulence of Histophilus somni. Trends Microbiol. 2010 Feb;18(2):90-9.
- Sandal I, Inzana TJ, Corbeil L. Haemophilus. In Pathogenesis of bacterial infections in animals, 4th Edition, C.L. Gyles, J.F. Prescott, G. Songer, C. Thoen (eds). Wiley/Blackwell, Ames, Iowa. 2010; Chapter 20, p. 387-409.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Histophilus somni is an etiologic agent of bronchopneumonia and systemic disease in bovines, and can form biofilms in vitro and in vivo. Thus, formation of a biofilm may be crucial to the persistence of H. somni in vivo. Microscopic analysis was used to confirm that H. somni was capable of forming a biofilm in cardiopulmonary tissue of the bovine host following respiratory challenge. The addition of N-acetylneuraminic acid (sialic acid) to media increased biofilm production by strain 2336 (a pneumonia isolate), but not by strain 129Pt (a preputial commensal isolate), as determined by crystal violet assay and scanning electron microscopy. Mutants of Histophilus somni were generated using the EZ::Tn5TM Tnp Transposome (Epicentre), and screened for altered biofilm formation. Twenty-five mutants were identified that produced 50-70% less biofilm than the parent strain, which was confirmed by scanning electron microscopy. The transposon (Tn) insertion sites of these biofilm-defective mutants were identified by sequencing of inverse PCR products. Some biofilm-deficient mutants had the Tn located in different regions of a gene encoding for filamentous hemagglutinin (FhaB), and the outer membrane transporter involved in the secretion of FhaB (FhaC). Other mutants had mutations in a gene encoding for an outer membrane efflux protein (TolC), which aids in intracellular trafficking and secretion. Some mutants that formed a diminished biofilm also produced less exopolysaccharide (EPS), which was determined by the amount of EPS that could be isolated. The putative genes that encode this EPS have also been determined to be upregulated during biofilm formation, but not during planktonic growth. The structure of the EPS was determined, and it has been identified as a galacto-mannan polymer with an alpha-(1-6)-linked, highly branched, comb-like mannopyranan polysaccharide structure with mannopyranose (Manp) units branched at C-2, which is similar in structure to that of yeast mannan. The virulence and serum resistance of these mutants in a mouse model is currently being examined, and if attenuated, will be tested further for biofilm formation and virulence in cattle. We also determined that the H. somni genome is over-represented in Haemophilus influenzae uptake signal sequences, and that the genome contains all the genes required for natural competence with the exception of comD. Using a modification of the standard transformation protocol employed for H. influenzae, H. somni strains 2336 and129Pt were successfully transformed with homologous DNA containing a kanamycin resistance gene as a marker. Therefore, transposon mutagenesis and natural transformation may prove to be efficient methods of mutagenesis for H. somni. PARTICIPANTS: PI-Thomas J Inzana, overall responsibility for the design and execution of experiments. Responsible for reports and for communication on all manuscripts. Co-PI - Indra Sandal, responsible for the execution of the experiments and immediate supervision of graduate students working on the project. Graduate Student - During this reporting period Yu Pan was supported on this project to determine if genes that affected biofilm formation contributed to virulence. Partner organization and collaborators included the Virginia Bioinformatics Institute and Oswald Crasta, who contributed to identifying the sites and sequences of genes containing transposon mutations. Graduate students Yu Pan and Mohamed El-Hadidy (not paid on the grant), and several undergraduate students (not paid on the grant) received training in transposon mutagenesis, sequencing, and biofilm formation from assisting on this project. TARGET AUDIENCES: Animal health pharmaceutical companies, bovine farmers, bovine veterinarians, microbiologists, and infectious disease researchers could all benefit from the information generated from this project. The information generated from this research would be of benefit in the classroom, particularly at the graduate level and senior undergraduate level. The information would also benefit those interested in biofilm research, Histophilus somni, and vaccine development. PROJECT MODIFICATIONS: A graduate student initially recruited to work on this project left the program after the first year, and a new student is only now able to be recruited. The availability of our bovine practitioner to work with us on this project and to get cattle for the research has been delayed due to scheduling conflicts and availability of animals free of antibodies to H. somni.
Impacts
The results of this work indicate that the formation of a biofilm by H. somni is an important virulence factor and may be a requirement for this organism to cause disease. Multiple genes have been identified that contribute to biofilm formation, some of which are absent in a commensal strain unable to cause disease. Data supporting that an exopolysaccharide is only formed during biofilm formation, and may be useful in diagnostic tests to differentiate infected cattle from those only exposed to or carrying the agent harmlessly. An improved method for mutagenesis has been identified and applied to isolating mutants that are deficient in biofilm formation. The results of this work will be important for the development of new, more effective vaccines and diagnostic tests for this pathogen, and in determining the importance and role of a biofilm is disease due to H. somni. Furthermore, this work shows that H. somni grown in the laboratory in or on culture media are very different from the bacteria that grow in the host, and investigators must be very careful in the interpretation of such laboratory results in light of in vivo pathogenesis.
Publications
- Inzana, T.J., W. E. Swords, I. Sandal, and S. Siddaramappa. 2008. Lipopolysaccharides, Biofilms, and Quorum Sensing in the Pasteurellaceae. pp. 177-196. In: Pasteurellaceae: Biology, Genomics and Molecular Aspects. Eds. P. Kuhnert and H. Christensen. Horizon Scientific Press. Hethersett, Norwich, UK.
- Sandal, I., M.N. Seleem, S.F. Elswaifi, N. Sriranganathan, and T.J. Inzana. 2008. Construction of a high-efficiency shuttle vector for Histophilus somni. J. Microbiol. Methods. 74:106-109.
- Sandal, I., J.Q. Shao, S. Annadata, M.A. Apicella, M. Boye, T.K. Jensen, G.K. Saunders, and T.J. Inzana. 2009. Histophilus somni biofilm formation in cardiopulmonary tissue of the bovine host following respiratory challenge. Microbes Infect. 11:254-263.
- Sandal, I. and T.J. Inzana. 2009. A genomic window into the virulence of Histophilus somni. Trends Microbiol. In press.
- Sandal I, Inzana TJ, Corbeil L. 2009. Haemophilus, In J. F. Prescott C. L. Gyles, J. G. Songer, and C. O. Thoen. (ed.), Pathogenesis of bacterial infections in animals, 4th ed. Blackwell Publishing Ltd., Oxford. In press.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: H. somni is an etiologic agent of bronchopneumonia and systemic disease in bovines, and can form biofilms in vitro and in vivo. Thus, formation of a biofilm may be crucial to the persistence of H. somni in vivo. Microscopic analyses were used to confirm that H. somni was capable of forming a biofilm in cardiopulmonary tissue of the bovine host following respiratory challenge. The addition of N-acetylneuraminic acid (sialic acid) to media increased biofilm production by strain 2336 (a pneumonia isolate), whereas biofilm formation by strain 129Pt (a preputial commensal isolate) was unchanged (determined by crystal violet assay and scanning electron microscopy). Electrospray mass spectrometry analysis of lipooligosaccharide (LOS) isolated from a biofilm, planktonic phase bacteria, and plate-grown bacteria of both strains 2336 and 129Pt showed that the degree of sialylation for several sialylated LOS glycoforms was unchanged during biofilm or planktonic growth. However, the exopolysaccharide (EPS) from a biofilm of strain 2336 supplemented with sialic acid was sialylated, but the EPS from strain 129Pt was not. Furthermore, two aminosugars (GlcNAc and GalNAc) were identified, in addition to the mannose, glucose and galactose normally present in the EPS of biofilms from strains 129Pt and 2336 grown without sialic acid. In order to identify genes involved in biofilm formation, the commercial Tn5 transposon EZ::Tn5Tnp Transposome (Epicentre) was used to isolate mutants altered in biofilm formation, as determined by crystal violet assay and electron microscopy. Random amplification of transposon ends (RATE) and sequencing of these RATE products was used to identify the genes mutated. Some mutants deficient in biofilm formation had an insertion in a homolog of the filamentous haemagglutinin (FHA), predicted to be an attachment factor. In vitro comparative gene expression analysis was carried out at 3, 5, and 7days of biofilm growth and of planktonic cell growth to identify genes that contribute to biofilm formation. Thirty genes were selected, from which 19 genes were from the putative EPS locus that we hypothesize to be involved in biofilm formation. An 8-fold increase in gene expression was observed in an ABC-type sugar transport system (rbs2a) during biofilm formation by strain 2336. Fourteen other genes of the EPS locus were also significantly upregulated in strain 2336 biofilm compared to planktonic cells, whereas in strain 129Pt only five EPS genes were upregulated. In addition, genes associated with LOS biosynthesis (siaB and licD) were also upregulated. These results provide new insights at the molecular level regarding gene expression by H. somni grown under biofilm conditions, and will allow better understanding of the mechanism and importance of biofilm formation in disease sites of the host. This work will be presented at the 2008 Animal Protection PD meeting in Chicago, IL December 7, 2008 and at the 2008 Conference for Research Workers in Animal Diseases December 10, 2008. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The results of this work indicate that the formation of a biofilm by H. somni is an important virulence factor and may be a requirement for this organism to cause disease. Multiple genes have been identified that contribute to biofilm formation, many of which are absent in a commensal strain unable to cause disease. The results of this work will be important for the development of new, more effective vaccines and diagnostic tests for this pathogen. Furthermore, this work shows that H. somni grown in the laboratory in or on culture media are very different from the bacteria that grow in the host, and investigators must be very careful in the interpretation of such laboratory results in light of in vivo pathogenesis.
Publications
- No publications reported this period
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