Anne M Wertheimer
- Assistant Research Professor
- Assistant Professor, Applied BioSciences - GIDP
- Immunization Champions and Mentors Program
- Gerontological Society of America, Fall 2015
- Butler Williams Summer NIA Program
- National Institute on Aging, Summer 2015
No activities entered.
Internship in Applied BiosciABS 593A (Spring 2018)
Master's ReportABS 909 (Spring 2018)
Reg Issues in Lab MgmntGRAD 595D (Spring 2018)
Independent StudyABS 599 (Fall 2017)
Internship in Applied BiosciABS 593A (Fall 2017)
Master's ReportABS 909 (Fall 2017)
Internship in Applied BiosciABS 593A (Summer I 2017)
Master's ReportABS 909 (Summer I 2017)
Internship in Applied BiosciABS 593A (Spring 2017)
Master's ReportABS 909 (Spring 2017)
Reg Issues in Lab MgmntGRAD 595D (Spring 2017)
Internship in Applied BiosciABS 593A (Fall 2016)
Master's ReportABS 909 (Fall 2016)
Internship in Applied BiosciABS 593A (Summer I 2016)
Master's ReportABS 909 (Summer I 2016)
- Nikolich-Žugich, J., Goldman, D. P., Cohen, P. R., Cortese, D., Fontana, L., Kennedy, B. K., Mohler, M. J., Olshansky, S. J., Perls, T., Perry, D., Richardson, A., Ritchie, C., Wertheimer, A. M., Faragher, R. G., & Fain, M. J. (2016). Preparing for an Aging World: Engaging Biogerontologists, Geriatricians, and the Society. The journals of gerontology. Series A, Biological sciences and medical sciences, 71(4), 435-44.More infoAlthough the demographic revolution has produced hundreds of millions people aged 65 and older, a substantial segment of that population is not enjoying the benefits of extended healthspan. Many live with multiple chronic conditions and disabilities that erode the quality of life. The consequences are also costly for society. In the United States, the most costly 5% of Medicare beneficiaries account for approximately 50% of Medicare's expenditures. This perspective summarizes a recent workshop on biomedical approaches to best extend healthspan as way to reduce age-related dysfunction and disability. We further specify the action items necessary to unite health professionals, scientists, and the society to partner around the exciting and palpable opportunities to extend healthspan.
- Pulko, V., Davies, J. S., Martinez, C., Lanteri, M. C., Busch, M. P., Diamond, M. S., Knox, K., Bush, E. C., Sims, P. A., Sinari, S., Billheimer, D., Haddad, E. K., Murray, K. O., Wertheimer, A. M., & Nikolich-Žugich, J. (2016). Human memory T cells with a naive phenotype accumulate with aging and respond to persistent viruses. Nature immunology, 17(8), 966-75.More infoThe number of naive T cells decreases and susceptibility to new microbial infections increases with age. Here we describe a previously unknown subset of phenotypically naive human CD8(+) T cells that rapidly secreted multiple cytokines in response to persistent viral antigens but differed transcriptionally from memory and effector T cells. The frequency of these CD8(+) T cells, called 'memory T cells with a naive phenotype' (TMNP cells), increased with age and after severe acute infection and inversely correlated with the residual capacity of the immune system to respond to new infections with age. CD8(+) TMNP cells represent a potential new target for the immunotherapy of persistent infections and should be accounted for and subtracted from the naive pool if truly naive T cells are needed to respond to antigens.
- Metcalf, T. U., Cubas, R. A., Ghneim, K., Cartwright, M. J., Grevenynghe, J. V., Richner, J. M., Olagnier, D. P., Wilkinson, P. A., Cameron, M. J., Park, B. S., Hiscott, J. B., Diamond, M. S., Wertheimer, A. M., Nikolich-Zugich, J., & Haddad, E. K. (2015). Global analyses revealed age-related alterations in innate immune responses after stimulation of pathogen recognition receptors. Aging cell, 14(3), 421-32.More infoAging leads to dysregulation of multiple components of the immune system that results in increased susceptibility to infections and poor response to vaccines in the aging population. The dysfunctions of adaptive B and T cells are well documented, but the effect of aging on innate immunity remains incompletely understood. Using a heterogeneous population of peripheral blood mononuclear cells (PBMCs), we first undertook transcriptional profiling and found that PBMCs isolated from old individuals (≥ 65 years) exhibited a delayed and altered response to stimulation with TLR4, TLR7/8, and RIG-I agonists compared to cells obtained from adults (≤ 40 years). This delayed response to innate immune agonists resulted in the reduced production of pro-inflammatory and antiviral cytokines and chemokines including TNFα, IL-6, IL-1β, IFNα, IFNγ, CCL2, and CCL7. While the major monocyte and dendritic cell subsets did not change numerically with aging, activation of specific cell types was altered. PBMCs from old subjects also had a lower frequency of CD40+ monocytes, impaired up-regulation of PD-L1 on monocytes and T cells, and increased expression of PD-L2 and B7-H4 on B cells. The defective immune response to innate agonists adversely affected adaptive immunity as TLR-stimulated PBMCs (minus CD3 T cells) from old subjects elicited significantly lower levels of adult T-cell proliferation than those from adult subjects in an allogeneic mixed lymphocyte reaction (MLR). Collectively, these age-associated changes in cytokine, chemokine and interferon production, as well as co-stimulatory protein expression could contribute to the blunted memory B- and T-cell immune responses to vaccines and infections.
- Mohler, M. J., Fain, M. J., Wertheimer, A. M., Najafi, B., & Nikolich-Žugich, J. (2014). The Frailty syndrome: clinical measurements and basic underpinnings in humans and animals. Experimental gerontology, 54, 6-13.More infoFrailty is an increasingly recognized syndrome resulting in age-related decline in function and reserve across multiple physiologic systems. It presents as a hyperinflammable state, characterized by high vulnerability for adverse health outcomes, such as disability, falls, hospitalization, institutionalization, and mortality. The prevalence of Frailty Syndrome (FS) is of potentially enormous significance, as it potentially affects 20-30% of adults older than 75. Cellular and molecular basis of frailty has not been elucidated. The objective of this review is to discuss recent advances in: (i) the potential cellular and molecular basis of Frailty Syndrome, including development of new models to study it; (ii) the human and animal measures of Frailty Syndrome; and (iii) the development of objective cross-species correlates to aid the basic understanding, diagnosis, treatment and rehabilitation of Frailty Syndrome in older adults.
- Wertheimer, A. M., Bennett, M. S., Park, B., Uhrlaub, J. L., Martinez, C., Pulko, V., Currier, N. L., Nikolich-Žugich, D., Kaye, J., & Nikolich-Žugich, J. (2014). Aging and cytomegalovirus infection differentially and jointly affect distinct circulating T cell subsets in humans. Journal of immunology (Baltimore, Md. : 1950), 192(5), 2143-55.More infoThe impact of intrinsic aging upon human peripheral blood T cell subsets remains incompletely quantified and understood. This impact must be distinguished from the influence of latent persistent microorganisms, particularly CMV, which has been associated with age-related changes in the T cell pool. In a cross-sectional cohort of 152 CMV-negative individuals, aged 21-101 y, we found that aging correlated strictly to an absolute loss of naive CD8, but not CD4, T cells but, contrary to many reports, did not lead to an increase in memory T cell numbers. The loss of naive CD8 T cells was not altered by CMV in 239 subjects (range 21-96 y), but the decline in CD4(+) naive cells showed significance in CMV(+) individuals. These individuals also exhibited an absolute increase in the effector/effector memory CD4(+) and CD8(+) cells with age. That increase was seen mainly, if not exclusively, in older subjects with elevated anti-CMV Ab titers, suggesting that efficacy of viral control over time may determine the magnitude of CMV impact upon T cell memory, and perhaps upon immune defense. These findings provide important new insights into the age-related changes in the peripheral blood pool of older adults, demonstrating that aging and CMV exert both distinct and joint influence upon blood T cell homeostasis in humans.
- Griffiths, S. J., Riddell, N. E., Masters, J., Libri, V., Henson, S. M., Wertheimer, A., Wallace, D., Sims, S., Rivino, L., Larbi, A., Kemeny, D. M., Nikolich-Zugich, J., Kern, F., Klenerman, P., Emery, V. C., & Akbar, A. N. (2013). Age-associated increase of low-avidity cytomegalovirus-specific CD8+ T cells that re-express CD45RA. Journal of immunology (Baltimore, Md. : 1950), 190(11), 5363-72.More infoThe mechanisms regulating memory CD8(+) T cell function and homeostasis during aging are unclear. CD8(+) effector memory T cells that re-express CD45RA increase considerably in older humans and both aging and persistent CMV infection are independent factors in this process. We used MHC class I tetrameric complexes that were mutated in the CD8 binding domain to identify CMV-specific CD8(+) T cells with high Ag-binding avidity. In individuals who were HLA-A*0201, CD8(+) T cells that expressed CD45RA and were specific for the pp65 protein (NLVPMVATV epitope) had lower avidity than those that expressed CD45RO and demonstrated decreased cytokine secretion and cytolytic potential after specific activation. Furthermore, low avidity NLVPMVATV-specific CD8(+) T cells were significantly increased in older individuals. The stimulation of blood leukocytes with CMV lysate induced high levels of IFN-α that in turn induced IL-15 production. Moreover, the addition of IL-15 to CD45RA(-)CD45RO(+) CMV-specific CD8(+) T cells induced CD45RA expression while Ag activated cells remained CD45RO(+). This raises the possibility that non-specific cytokine-driven accumulation of CMV-specific CD8(+)CD45RA(+) T cells with lower Ag-binding avidity may exacerbate the effects of viral reactivation on skewing the T cell repertoire in CMV-infected individuals during aging.
- Weyand, N. J., Wertheimer, A. M., Hobbs, T. R., Sisko, J. L., Taku, N. A., Gregston, L. D., Clary, S., Higashi, D. L., Biais, N., Brown, L. M., Planer, S. L., Legasse, A. W., Axthelm, M. K., Wong, S. W., & So, M. (2013). Neisseria infection of rhesus macaques as a model to study colonization, transmission, persistence, and horizontal gene transfer. Proceedings of the National Academy of Sciences of the United States of America, 110(8), 3059-64.More infoThe strict tropism of many pathogens for man hampers the development of animal models that recapitulate important microbe-host interactions. We developed a rhesus macaque model for studying Neisseria-host interactions using Neisseria species indigenous to the animal. We report that Neisseria are common inhabitants of the rhesus macaque. Neisseria isolated from the rhesus macaque recolonize animals after laboratory passage, persist in the animals for at least 72 d, and are transmitted between animals. Neisseria are naturally competent and acquire genetic markers from each other in vivo, in the absence of selection, within 44 d after colonization. Neisseria macacae encodes orthologs of known or presumed virulence factors of human-adapted Neisseria, as well as current or candidate vaccine antigens. We conclude that the rhesus macaque model will allow studies of the molecular mechanisms of Neisseria colonization, transmission, persistence, and horizontal gene transfer. The model can potentially be developed further for preclinical testing of vaccine candidates.
- Wertheimer, A. M., Uhrlaub, J. L., Hirsch, A., Medigeshi, G., Sprague, J., Legasse, A., Wilk, J., Wiley, C. A., Didier, P., Tesh, R. B., Murray, K. O., Axthelm, M. K., Wong, S. W., & Nikolich-Žugich, J. (2010). Immune response to the West Nile virus in aged non-human primates. PloS one, 5(12), e15514.More infoRisk of encephalitis from West Nile virus (WNV) infection increases dramatically with age. Understanding the basis of this susceptibility requires development of suitable animal models. Here, we investigated the immune response to WNV in old non-human primates.
- Lee, J. A., Spidlen, J., Boyce, K., Cai, J., Crosbie, N., Dalphin, M., Furlong, J., Gasparetto, M., Goldberg, M., Goralczyk, E. M., Hyun, B., Jansen, K., Kollmann, T., Kong, M., Leif, R., McWeeney, S., Moloshok, T. D., Moore, W., Nolan, G., , Nolan, J., et al. (2008). MIFlowCyt: the minimum information about a Flow Cytometry Experiment. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 73(10), 926-30.More infoA fundamental tenet of scientific research is that published results are open to independent validation and refutation. Minimum data standards aid data providers, users, and publishers by providing a specification of what is required to unambiguously interpret experimental findings. Here, we present the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, stating the minimum information required to report flow cytometry (FCM) experiments. We brought together a cross-disciplinary international collaborative group of bioinformaticians, computational statisticians, software developers, instrument manufacturers, and clinical and basic research scientists to develop the standard. The standard was subsequently vetted by the International Society for Advancement of Cytometry (ISAC) Data Standards Task Force, Standards Committee, membership, and Council. The MIFlowCyt standard includes recommendations about descriptions of the specimens and reagents included in the FCM experiment, the configuration of the instrument used to perform the assays, and the data processing approaches used to interpret the primary output data. MIFlowCyt has been adopted as a standard by ISAC, representing the FCM scientific community including scientists as well as software and hardware manufacturers. Adoptionof MIFlowCyt by the scientific and publishing communities will facilitate third-party understanding and reuse of FCM data.
- Wertheimer, A. M., Polyak, S. J., Leistikow, R., & Rosen, H. R. (2007). Engulfment of apoptotic cells expressing HCV proteins leads to differential chemokine expression and STAT signaling in human dendritic cells. Hepatology (Baltimore, Md.), 45(6), 1422-32.More infoIn the majority of cases, infection with hepatitis C virus (HCV) becomes chronic and is often associated with impaired innate and adaptive immune responses. The mechanisms underlying viral persistence and lack of protective immunity are poorly understood. Considering that dendritic cells (DCs) play critical roles in initiating and modulating immune responses, we explored the effect of HCV proteins on DC gene and protein expression, phenotype, and function. Human DCs were generated following plastic adherence of monocytes and culture with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) from normal subjects. Autologous nonadherent peripheral blood mononuclear cells were infected with vaccinia constructs expressing various HCV proteins (core-E1, NS5A, NS5B) or an irrelevant protein beta-galactosidase (beta-gal) as the control, induced to undergo apoptosis, then co-cultured with DCs. Between 2% and 10% of the genes probed in a cDNA nylon array were differentially regulated within DCs that had engulfed HCV proteins. In particular, the presence of intracellular NS5A led to increased transcriptional and protein expression of IL-8 (CXCL-8), a chemokine with proinflammatory and anti-interferon properties, and impaired interferon induction of signal transducers and activators of transcription 1 (STAT1) serine and tyrosine and STAT2 tyrosine phosphorylation.
- Tester, I., Smyk-Pearson, S., Wang, P., Wertheimer, A., Yao, E., Lewinsohn, D. M., Tavis, J. E., & Rosen, H. R. (2005). Immune evasion versus recovery after acute hepatitis C virus infection from a shared source. The Journal of experimental medicine, 201(11), 1725-31.More infoAcute infection with hepatitis C virus (HCV) rarely is identified, and hence, the determinants of spontaneous resolution versus chronicity remain incompletely understood. In particular, because of the retrospective nature and unknown source of infection in most human studies, direct evidence for emergence of escape mutations in immunodominant major histocompatibility complex class I-restricted epitopes leading to immune evasion is extremely limited. In two patients infected accidentally with an identical HCV strain but who developed divergent outcomes, the total lack of HCV-specific CD4+ T cells in conjunction with vigorous CD8+ T cells that targeted a single epitope in one patient was associated with mutational escape and viral persistence. Statistical evidence for positive Darwinian selective pressure against an immunodominant epitope is presented. Wild-type cytotoxic T lymphocytes persisted even after the cognate antigen was no longer present.
- Weston, S. J., Leistikow, R. L., Reddy, K. R., Torres, M., Wertheimer, A. M., Lewinsohn, D. M., Chou, S., Davey, M. P., Corless, C., O'Farrelly, C., Nelson, D. R., & Rosen, H. R. (2005). Reconstitution of hepatitis C virus-specific T-cellmediated immunity after liver transplantation. Hepatology (Baltimore, Md.), 41(1), 72-81.More infoHepatitis C virus (HCV)-related liver failure is the leading indication for liver transplantation worldwide. After transplantation, virological recurrence is the rule, but the spectrum of histological injury is wide, ranging from the development of allograft cirrhosis within a few years to minimal hepatitis despite long-term follow-up. The immunological correlates of this variable natural history are poorly understood. Here, we studied the kinetics of the cellular immune responses, viral replication, and allograft histology in 24 patients who had undergone liver transplantation for HCV-related liver failure. Using direct ex vivo methodologies (i.e., interferon-gamma ELISPOT and major histocompatibility complex class I-peptide tetrameric complexes), we found that patients who experienced viral eradication after antiviral therapy showed restoration of HCV-specific T-cell responses, whereas patients with progressive HCV recurrence that failed to respond to therapy showed declining frequencies of these viral-specific effector cells. The cytotoxic T lymphocytes that peripherally reconstituted after transplantation were clonotypically identical to those present within the recipient explant liver, defined at the level of the T-cell receptor beta chain (one epitope/one clone). Moreover, the subset of patients who spontaneously demonstrated minimal histologic recurrence had more vigorous CD4+ T-cell responses in the first 3 months, particularly targeting nonstructural proteins. We provide evidence that T-cell responses emerge after liver transplantation, and their presence correlates with improved histological and clinical outcomes. In conclusion, these results may help identify patients more likely to develop severe HCV recurrence and therefore benefit from current antiviral therapy, as well as provide a rationale for the future use of novel immunotherapeutic approaches. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience. wiley.com/jpages/0270-9139/suppmat/index.html).
- Rosen, H. R., Hinrichs, D. J., Leistikow, R. L., Callender, G., Wertheimer, A. M., Nishimura, M. I., & Lewinsohn, D. M. (2004). Cutting edge: identification of hepatitis C virus-specific CD8+ T cells restricted by donor HLA alleles following liver transplantation. Journal of immunology (Baltimore, Md. : 1950), 173(9), 5355-9.More infoBy necessity, human liver transplantation is performed across HLA barriers. As a result, intracellular infection of the allograft presents a unique immunologic challenge for the recipient's immune system. In this study, we describe the presence of HLA-A2-restricted, hepatitis C virus (HCV)-specific CD8+ T cells in liver transplant recipients in whom the allograft is HLA-A2 positive and the recipient is HLA-A2 negative. These memory-effector T cells are recipient derived and recognize HCV peptide uniquely in the context of HLA-A2. Furthermore, these cells were absent before the transplant, suggesting that the allograft is capable of selectively expanding naive CD8+ T cells. The in vitro specificity to donor HLA allele-restricted CD8+ T cells suggests that these cells may function to control HCV spread in the allograft.
- Wertheimer, A. M., Bakke, A., & Rosen, H. R. (2004). Direct enumeration and functional assessment of circulating dendritic cells in patients with liver disease. Hepatology (Baltimore, Md.), 40(2), 335-45.More infoChronic liver disease has been shown to be associated with diminished humoral and cellular immune function. Although antigen-presenting cells (APC) that initiate immune responses include various cells (B cells, endothelial cells, macrophages, etc.), the dendritic cell (DC) is a professional APC that activates naive T cells most efficiently. To examine the frequency and function of DCs in chronic liver disease, we studied circulating DCs from a cohort of 112 subjects (23 normal subjects, 29 subjects who had spontaneously recovered from hepatitis C virus [HCV] infection, 30 chronically infected HCV patients, and 30 patients with liver disease unrelated to HCV infection). Our analyses revealed significant reduction in both circulating myeloid (mDC) and plasmacytoid dendritic cells (pDC) in patients with liver disease. In contrast, examination of subjects with spontaneously resolved HCV infection revealed no significant difference in either circulating mDCs or pDCs. We found an inverse correlation with serum alanine aminotransferase (ALT) levels and both mDCs and pDCs frequency. In a subset of patients for whom intrahepatic cells were available, paired analysis revealed enrichment for DCs within the intrahepatic compartment. Interferon alfa (IFN-alpha) production in response to influenza A and poly (I:C) correlated with the frequency of circulating DCs, although IFN-alpha production was comparable on a per-DC basis in patients with liver disease. In conclusion, patients with liver disease exhibit a reduction in circulating DCs. Considering that DCs are essential for initiation and regulation of innate and adaptive immunity, these findings have implications for both viral persistence and liver disease.
- Di Lorenzo, M., Stork, M., Tolmasky, M. E., Actis, L. A., Farrell, D., Welch, T. J., Crosa, L. M., Wertheimer, A. M., Chen, Q., Salinas, P., Waldbeser, L., & Crosa, J. H. (2003). Complete sequence of virulence plasmid pJM1 from the marine fish pathogen Vibrio anguillarum strain 775. Journal of bacteriology, 185(19), 5822-30.More infoThe virulence plasmid pJM1 enables the fish pathogen Vibrio anguillarum, a gram-negative polarly flagellated comma-shaped rod bacterium, to cause a highly fatal hemorrhagic septicemic disease in salmonids and other fishes, leading to epizootics throughout the world. The pJM1 plasmid 65,009-nucleotide sequence, with an overall G+C content of 42.6%, revealed genes and open reading frames (ORFs) encoding iron transporters, nonribosomal peptide enzymes, and other proteins essential for the biosynthesis of the siderophore anguibactin. Of the 59 ORFs, approximately 32% were related to iron metabolic functions. The plasmid pJM1 confers on V. anguillarum the ability to take up ferric iron as a complex with anguibactin from a medium in which iron is chelated by transferrin, ethylenediamine-di(o-hydroxyphenyl-acetic acid), or other iron-chelating compounds. The fatDCBA-angRT operon as well as other downstream biosynthetic genes is bracketed by the homologous ISV-A1 and ISV-A2 insertion sequences. Other clusters on the plasmid also show an insertion element-flanked organization, including ORFs homologous to genes involved in the biosynthesis of 2,3-dihydroxybenzoic acid. Homologues of replication and partition genes are also identified on pJM1 adjacent to this region. ORFs with no known function represent approximately 30% of the pJM1 sequence. The insertion sequence elements in the composite transposon-like structures, corroborated by the G+C content of the pJM1 sequence, suggest a modular composition of plasmid pJM1, biased towards acquisition of modules containing genes related to iron metabolic functions. We also show that there is considerable microheterogeneity in pJM1-like plasmids from virulent strains of V. anguillarum isolated from different geographical sources.
- Wertheimer, A. M., Miner, C., Lewinsohn, D. M., Sasaki, A. W., Kaufman, E., & Rosen, H. R. (2003). Novel CD4+ and CD8+ T-cell determinants within the NS3 protein in subjects with spontaneously resolved HCV infection. Hepatology (Baltimore, Md.), 37(3), 577-89.More infoSpontaneous resolution of hepatitis C virus (HCV) infection is a relatively infrequent event, and these individuals provide a unique opportunity to characterize correlates of protective immunity as an important first step in the development of vaccine candidates. The aim of this study was to directly and comprehensively enumerate HCV-nonstructural protein 3 (NS3) specific CD4(+) and CD8(+) T cells ex vivo from HLA diverse individuals who had been successful in spontaneously resolving HCV infection. We measured interferon gamma (IFN-gamma) production with an ELISPOT assay using magnetic bead-separated CD4(+) or CD8(+) T cells in response to autologous DCs that had been pulsed with 15mer per peptides overlapping by 11 amino acids and spanning all of the NS3 protein (150 total peptides). All subjects with spontaneously recovered HCV infection demonstrated vigorous and multispecific CD4(+) T-cell responses to NS3 peptides, and 6 of 10 subjects demonstrated CD8(+) T-cell responses. More importantly, we identified novel, previously unpredicted antigenic regions, which in most cases elicited high frequencies within a given individual. In conclusion, subjects who have spontaneously eradicated HCV infection up to 35 years earlier demonstrate persistent CD4(+) and CD8(+) T-cell responses specific to NS3. By providing a comprehensive screening of all potential T-cell epitopes contained in the NS3 region, our strategy defines the breadth of the T-cell response and identifies novel, unpredicted specificities.
- Wertheimer, A. M., Verweij, W., Chen, Q., Crosa, L. M., Nagasawa, M., Tolmasky, M. E., Actis, L. A., & Crosa, J. H. (1999). Characterization of the angR gene of Vibrio anguillarum: essential role in virulence. Infection and immunity, 67(12), 6496-509.More infoThe ability to utilize the iron bound by high-affinity iron-binding proteins in the vertebrate host is an important virulence factor for the marine fish pathogen Vibrio anguillarum. Virulence in septicemic infections is due to the presence of a highly efficient plasmid-encoded iron transport system. AngR, a 110-kDa protein component of this system, appears to play a role in both regulation of the expression of the iron transport genes fatDCBA and the production of the siderophore anguibactin. Therefore, study of the expression of the angR gene and the properties of its product, the AngR protein, may contribute to the understanding of the mechanisms of virulence of this pathogen. In this work, we present genetic and molecular evidence from transposition mutagenesis experiments and RNA analysis that angR, which maps immediately downstream of the fatA gene, is part of a polycistronic transcript that also includes the iron transport genes fatDCBA and angT, a gene located downstream of angR which showed domain homology to certain thioesterases involved in nonribosomal peptide synthesis of siderophores and antibiotics. In order to dissect the specific domains of AngR associated with regulation of iron transport gene expression, anguibactin production, and virulence, we also generated a panel of site-directed angR mutants, as well as deletion derivatives. Both virulence and anguibactin production were dramatically affected by each one of the angR modifications. In contrast to the need for an intact AngR molecule for anguibactin production and virulence, the regulation of iron transport gene expression does not require the entire AngR molecule, since truncation of the carboxy terminus carrying the nonribosomal peptide synthetase cores, as well as the site-directed mutations, resulted in derivatives that retained their ability to regulate gene expression which was only abolished after truncation of amino-terminal sequences containing helix-turn-helix and leucine zipper motifs and a specialized heterocyclization and condensation domain found in certain nonribosomal peptide synthetases. The evidence, while not rigorously eliminating the possibility that a separate regulatory polypeptide exists and is encoded somewhere within the 5'-end region of the angR gene, strongly supports the idea that AngR is a bifunctional protein and that it plays an essential role in the virulence mechanisms of V. anguillarum. We also show in this study that the angT gene, found downstream of angR, intervenes in the mechanism of anguibactin production but is not essential for virulence or iron transport gene expression.
- Chen, Q., Wertheimer, A. M., Tolmasky, M. E., & Crosa, J. H. (1996). The AngR protein and the siderophore anguibactin positively regulate the expression of iron-transport genes in Vibrio anguillarum. Molecular microbiology, 22(1), 127-34.More infoVibrio anguillarum virulence is associated with the presence of a plasmid-mediated iron-uptake system expressed under iron-limiting conditions, which consists of the siderophore anguibactin and specific iron-transport proteins. This system is maximally expressed under iron-limiting conditions and requires the AngR protein that acts as a positive regulator of anguibactin biosynthesis and also possess an EntE-like enzymatic function that may play a role in anguibactin biosynthesis. In this work, we demonstrate that in addition to possessing these functions related to anguibactin production, AngR also positively regulated transcription of the iron-transport genes fatA and fatB. We also show that transcription of angR is repressed by Fur under iron-rich conditions. In addition, we present evidence that anguibactin itself enhanced transcription of the iron-transport genes fatA and fatB, independently of AngR and the trans-acting factor (TAF) product(s). The presence of either AngR (together with the TAF product(s)) or anguibactin alone led to a partial level of expression of the iron-transport genes fatA and fatB, while full expression is achieved when AngR, the TAF products and anguibactin are all present.
- Tolmasky, M. E., Wertheimer, A. M., Actis, L. A., & Crosa, J. H. (1994). Characterization of the Vibrio anguillarum fur gene: role in regulation of expression of the FatA outer membrane protein and catechols. Journal of bacteriology, 176(1), 213-20.More infoThe chromosomally encoded Vibrio anguillarum fur gene was characterized. The amino acid sequence of the Fur protein showed a very high degree of homology with those of V. cholerae and V. vulnificus. The degree of homology was lower, although still high, with the Escherichia coli and Yersinia pestis Fur amino acid sequences, while the lowest degree of homology was found with the Pseudomonas aeruginosa Fur protein. The C-terminal portion of Fur is the least conserved region among these Fur proteins. Within this portion, two regions spanning amino acids 105 to 121 and 132 to the end are the least conserved. A certain degree of variation is also present in the N termini spanning amino acids 28 to 46. Regulation of expression of the V. anguillarum fur gene by iron was not detected by immunoblot analysis. Mutations in the cloned fur gene were generated either by site-directed mutagenesis (the Lys-77 was changed to a Gly to generate the derivative FurG77) or by insertion of a DNA fragment harboring the aph gene in the same position. FurG77 was impaired in its ability to regulate a reporter gene with the Fur box in its promoter, while the insertion mutant was completely inactive. V. anguillarum fur mutants were obtained by isolating manganese-resistant derivatives. In one of these mutants, which encoded a Fur protein with an apparent lower molecular weight, the regulation of the production of catechols and synthesis of the outer membrane protein FatA were partially lost. In the case of another mutant, no protein was detected by anti-Fur serum. This derivative showed a total lack of regulation of biosynthesis of catechols and FatA protein by iron.
- Wertheimer, A. M., Tolmasky, M. E., Actis, L. A., & Crosa, J. H. (1994). Structural and functional analyses of mutant Fur proteins with impaired regulatory function. Journal of bacteriology, 176(16), 5116-22.More infoVibrio anguillarum Fur mutants, 775met9 and 775met11, were characterized. V. anguillarum 775met9 had a change of D to G at position 104 located in the carboxy terminus resulting in impaired Fur activity. Computer analysis predicts perturbation of an alpha-helix in the carboxy terminus which may interfere with Fur protein conformation. Strain 775met11 had a change in the start codon resulting in no protein synthesis. The mutants are unstable, and reversion to the wild type occurs frequently.