Virginia Miller, PhD

Virginia Miller, PhD

Professor
Department of Genetics
6202 Marsico Hall
CB#7290
919-966-9956



Research

Molecular and genetic analysis of virulence of Yersinia and Klebsiella: My laboratory uses Yersinia, and Klebsiella as model systems to study bacterial pathogenesis. The long-term goals of our work are to understand the bacteria-host interaction at the molecular level to learn how this interaction affects the pathogenesis of infections and to understand how these pathogens co-ordinate the expression of virulence determinants during an infection. To do this we use genetic, molecular and immunological approaches, in conjunction with the mouse model of infection. Some of our projects are outlined below.

RovA regulon of the Yersiniae

We have applied several genetic approaches to identify new virulence genes of Y. enterocolitica and are currently characterizing these genes, their products, and their role in disease. We also have been studying the invasion gene inv, with a focus on understanding the mechanism of regulation of expression of inv and the co-ordination of its expression with other virulence genes. An inv regulatory gene, rovA, has been identified that regulates expression of inv in the laboratory and during an infection. RovA also regulates expression of other novel virulence determinants that influence the early inflammatory response to Y. enterocolitica infection. Recently we extended the analysis of RovA to Yersinia pestis, the causative agent of bubonic and pneumonic plague and found that RovA is also required for full virulence of Y. pestis. Microarray analysis was used to identify the RovA regulated genes in Y. pestis and Y. enterocolitica. Sixty-four genes appeared to be RovA regulated in Y. enterocolitica and 73 in Y. pestis, suggesting the regulon may be quite large in each of these species. Our long-term goals are (i) to understand how these genes are regulated by RovA and how that is coordinated with expression of other virulence factors, and (ii) to determine which RovA regulated genes contribute to virulence and understand how they affect the host-pathogen interaction.

Ysa Type Three Secretion System of Y. enterocolitica

Type III secretion systems (T3SS) are a means by which Gram negative pathogens deliver effector proteins into host cells. One of the first and best, characterized systems is encoded on the virulence plasmid of the yersiniae. However, a second T3SS was recently identified on the chromosome of Y. enterocolitica (designated the Ysa T3SS). We identified some of the key players in the regulation of expression of this system and have begun to identify the effectors (designated Ysps) secreted by the system. The long-term goals are to understand the role of this system and the individual effectors in the biology of Y. enterocolitica as well as its regulation. However, the current focus of our research is on the regulation of this system. Our data indicates that the expression of the Ysa T3SS is dependent on a phosphorelay system with a number of unusual features. Typically in bacteria these phosphorelay systems are composed of two proteins (i.e. the two component regulatory system) in which one protein is the sensor (responds to an environmental signal) and the other the response regulator (usually a DNA binding protein). Some related but more complex systems (termed hybrid two component systems) have several transfers of phosphate in the sensor kinase component before the ultimate transfer to the response regulator by the histidine phosphotransferase (Hpt) domain of the sensor. What makes the regulation of the Ysa T3SS system unusual is that the Hpt “domain” (YsrT) is a separate, small protein rather than a domain of the sensor (YsrS). In addition, a second response regulator, RcsB, from another two-component system is required in addition to the response regulator YsrR for ysa expression, even though the sensor of the Rcs system is not required. How exactly these players are functioning to regulate the ysa/ysp system and the consequences of the unique regulatory set-up is the topic of ongoing research.

Dissecting early events in bubonic plague

Yersinia pestis is the causative agent of disease in a wide variety of mammals, and humans can become infected when human and animal ecologies intersect.  This has lead to three well recognized pandemics of plague in human history, and infection with Y. pestis is currently considered by the WHO as a re-emerging infectious disease because of the increase in incidence in a wide number of countries. Bubonic plague, the most common form of disease, occurs when an infected flea tries to feed on a susceptible host (including humans) and is characterized by the appearance of the bacteria in the lymph node (dLN) nearest to the bite site (inoculation site, IS).  Once Y. pestis reaches the dLN it is capable of replicating to high numbers and spreading systemically; disease progression to death is very rapid in susceptible hosts.  Transmission to a new host requires a systemic infection with high levels of bacteria in the blood (>107 cfu/ml) such that the flea will be colonized.  Thus, a key event for controlling infection with Y. pestis is to control dissemination from the IS to the dLN and dissemination of Y. pestis from the dLN to the blood.  It has long been believed that the bacteria disseminate to the dLN by trafficking in phagocytic cells from the IS via the lymphatic system.  However, there has been strikingly little data supporting this dogma. Due to the critical nature of these dissemination steps in the development of disease, it is a clear focus for vaccine or therapeutic targets, and yet early dissemination to the dLN, establishment of infection in the dLN and dissemination from the dLN are not well understood. 

 Using an intradermal route of inoculation (the route that most closely mimics that of the flea) and inoculation with a set of Y. pestis each marked with a unique oligonucleotide tag, we recently developed a dissemination assay (DA) to monitor the effectiveness of transit of bacteria from the IS to the dLN and to systemic tissues from the dLN.  This already has revealed new insights regarding dissemination of Y. pestis and raises new questions. Our recent results indicate there is a strong bottleneck between the IS and dLN, that neutrophils are not needed for trafficking to the dLN or for the bottleneck between the IS and dLN, and that the bacteria can disseminate as free bacteria in the lymphatics. Our long-term goal is to understand the early events ultimately leading to a successful systemic infection.  Specifically we propose to determine how known virulence factors affect specific steps between IS and blood, and how key host cells affect the development of pathology and systemic colonization.  Together these studies will give us a clearer picture of how host-pathogen interactions and specific virulence determinants affect development of bubonic plague, providing a foundation for development of intervention strategies.

Virulence factors of Klebsiella

For Klebsiella we have developed a mouse model of infection using an intranasal inoculation method. A bank of 5,000 transposon mutants have been isolated and screened in this intranasal model of infection for mutations that alter the ability of Klebsiella to either colonize the lung or spread from the lung to the spleen. A subset of these mutants is currently being studied in more detail. In particular we are interested in two mutants that identify a putative Type VI secretion system (T6SS) of Klebsiella. The T6SS have emerged in recent years as a new type of secretion system in Gram-negative bacteria and has been linked to virulence in both mammalian and plant pathogens.

Publications

Gonzalez, R. J., E. H. Weening, MC Lane, and V. L. Miller. 2015. Comparison of models for bubonic plague reveals unique pathogen adaptations to the dermis.  Infect Immun 83:2855-2861. (Recommended for F1000)

Pechous, R. D., C. A. Broberg, N. M. Stasulli, V. L. Miller and W. E. Goldman. 2015. In vivo transcriptional profiling of Yersinia pestis reveals a novel bacterial mediator of pulmonary inflammation.  Mbio 17: e02302-14.

Gonzalez, R. J., M. C. Lane, N. Wagner, E. H. Weening and V. L. Miller. 2015. Dissemination of a highly virulent pathogen: tracking the early events that define infection.  PLoS Pathog. 11:e1004587. [PMID: 25611317]

Broberg, C. A., Wu, W., Cavalcoli, J. D., Miller, V. L., and M. A. Bachman. 2014. Complete genome sequence of Klebsiella pneumoniae strain ATCC 43816 KPPR1, a rifampin-resistant mutant commonly used in animal, genetic, and molecular biology studies.  Genome Announc. 2:e00924. [PMIC25291761]

Wagner, N. J., C. P. Lin, L. B. Borst, and V. L. Miller. 2013.  YaxAB: a Yersinia enterocolitica pore-forming toxin regulated by RovA.  Infect. Immun. 81:4208-4219. (Epub September 3) [PMID: 24002058]

Lawrenz, M. B., J. Pennington, and V. L. Miller. 2013  Acquisition of omptin reveals cryptic virulence function of autotransporter YapE in Yersinia pestis. Mol. Microbiol.  89:276-287 (Epub May 23) [PMID: 23701256]

Lane, M. C., J. D. Lenz, and V. L. Miller. 2013. Proteolytic processing of the Yersinia pestis YapG autotransporter by the omptin protease Pla and the contribution of YapG to murine plague pathogenesis.  J. Med. Microbiol. 62:1124-1134 (Epub May 8) [PMID: 23657527]

Walker K. A., Maltez, V., Hall, J., Vitko, N., and V. L. Miller. 2013. A phenotype at last: Essential role for the Yersinia enterocolitica Ysa Type III secretion system in a Drosophila S2 cell model.  Infect. Immun. 81:2478-2487. (Epub April 29) [PMID: 23630961]

Broberg, C. A., Palacios, M., and V. L. Miller. 2013. Whole genome draft sequences of three multi-drug resistant Klebsiella pneumonia strains available from American Type Culture Collection. Genome Announcements 1:312-313. [PMID: PMC23723407]

Gonzalez, R. J., Weening, E. H., Frothingham, R., Sempowski, G. D., and V. L. Miller. 2012. Bioluminescence imaging to track bacterial dissemination of Yersinia pestis using different routes of infection in mice.  BMC Microbiol. 12:147(Epub July 24). [PMID: 22827851]

Lenz, J. D., Temple, B. R., and V. L. Miller. 2012.  Evolution and virulence contributions of the autotransporter proteins YapJ and YapK of Y. pestis CO92 and their homologs in Y. pseudotuberculosis IP32953.  Infect. Immun. 80:3693-3705 (Epub July 16) [PMID: 22802344]

Obrist, M. W., and V. L. Miller. 2012.  Low copy expression vectors for use in Yersinia sp. and related organisms.  Plasmid 68:33-42. [PMID: 22445322]

Lenz JD, Lawrenz MB, Cotter DG, Lane MC, Gonzalez RJ, Palacios M, Miller VL (2011). Expression during host infection and localization of Yersinia pestis autotransporter proteins (Yaps). J Bacteriol.

Weening EH, Cathelyn JS, Kaufman G, Lawrenz MB, Price P, Goldman WE, Miller VL (2011). The dependence of the Yersinia pestis capsule on pathogenesis is influenced by the mouse background.
Infect Immun. 79(2):644-52.

Walker KA, Obrist MW, Mildiner-Earley S, Miller VL (2010). Identification of YsrT and evidence that YsrRST constitute a unique phosphorelay system in Yersinia enterocolitica. J Bacteriol. 192(22):5887-97.

Walker KA, Miller VL (2009). Synchronous gene expression of the Yersinia enterocolitica Ysa type III secretion system and its effectors. J Bacteriol. 191(6):1816-26.

Lawrenz MB, Lenz JD, Miller VL 2009). A novel autotransporter adhesin is required for efficient colonization during bubonic plague. Infect Immun. 77(1):317-26.

Witowski SE, Walker KA, Miller VL (2008). YspM, a newly identified Ysa type III secreted protein of Yersinia enterocolitica. J Bacteriol. 190(22):7315-25.

Mildiner-Earley S, Walker KA, Miller VL (2007). Environmental stimuli affecting expression of the Ysa type three secretion locus. Adv Exp Med Biol. 603:211-6.

Cathelyn JS, Ellison DW, Hinchliffe SJ, Wren BW, Miller VL (2007). The RovA regulons of Yersinia enterocolitica and Yersinia pestis are distinct: evidence that many RovA-regulated genes were acquired more recently than the core genome. Mol Microbiol. 66(1):189-205.

Lawrenz MB, Miller VL (2007). Comparative analysis of the regulation of rovA from the pathogenic yersiniae. J Bacteriol. 189(16):5963-75.

Handley SA, Miller VL (2007). General and specific host responses to bacterial infection in Peyer's patches: a role for stromelysin-1 (matrix metalloproteinase-3) during Salmonella enterica infection. Mol Microbiol. 64(1):94-110.

Lawlor MS, O'connor C, Miller VL (2007). Yersiniabactin is a virulence factor for Klebsiella pneumoniae during pulmonary infection. Infect Immun. 75(3):1463-72.

Cathelyn JS, Crosby SD, Lathem WW, Goldman WE, Miller VL (2006). RovA, a global regulator of Yersinia pestis, specifically required for bubonic plague. Proc Natl Acad Sci U S A. 103(36):13514-9.

Lawlor MS, Handley SA, Miller VL (2006). Comparison of the host responses to wild-type and cpsB mutant Klebsiella pneumoniae infections. Infect Immun. 74(9):5402-7.

Ellison DW, Miller VL (2006). H-NS represses inv transcription in Yersinia enterocolitica through competition with RovA and interaction with YmoA. J Bacteriol. 188(14):5101-12.

Mildiner-Earley S, Miller VL (2006). Characterization of a novel porin involved in systemic Yersinia enterocolitica infection. Infect Immun. 74(7):4361-5.

Handley SA, Dube PH, Miller VL (2006). Histamine signaling through the H(2) receptor in the Peyer's patch is important for controlling Yersinia enterocolitica infection. Proc Natl Acad Sci U S A. 103(24):9268-73.

Handley SA, Newberry RD, Miller VL (2005). Yersinia enterocolitica invasin-dependent and invasin-independent mechanisms of systemic dissemination. Infect Immun. 73(12):8453-5.

Lawlor MS, Hsu J, Rick PD, Miller VL (2005). Identification of Klebsiella pneumoniae virulence determinants using an intranasal infection model. Mol Microbiol. 58(4):1054-73.

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