Bio

Honors & Awards


  • Walter V. and Idun Berry Postdoctoral Fellow, Stanford University (2019)

Professional Education


  • Doctor of Philosophy, Washington State University, Immunology & Infectious Diseases (2018)
  • Bachelor of Science, University of Washington (2011)

Stanford Advisors


Publications

All Publications


  • High-resolution mapping reveals that microniches in the gastric glands control Helicobacter pylori colonization of the stomach PLOS BIOLOGY Fung, C., Tan, S., Nakajima, M., Skoog, E. C., Camarillo-Guerrero, L., Klein, J. A., Lawley, T. D., Solnick, J. V., Fukami, T., Amieva, M. R. 2019; 17 (5)
  • Controlled Activity of the Salmonella Invasion-Associated Injectisome Reveals Its Intracellular Role in the Cytosolic Population. mBio Klein, J. A., Grenz, J. R., Slauch, J. M., Knodler, L. A. 2017; 8 (6)

    Abstract

    The Salmonella invasion-associated type III secretion system (T3SS1) is an essential virulence factor required for entry into nonphagocytic cells and consequent uptake into a Salmonella-containing vacuole (SCV). While Salmonella is typically regarded as a vacuolar pathogen, a subset of bacteria escape from the SCV in epithelial cells and eventually hyperreplicate in the cytosol. T3SS1 is downregulated following bacterial entry into mammalian cells, but cytosolic Salmonella cells are T3SS1 induced, suggesting prolonged or resurgent activity of T3SS1 in this population. In order to investigate the postinternalization contributions of T3SS1 to the Salmonella infectious cycle in epithelial cells, we bypassed its requirement for bacterial entry by tagging the T3SS1-energizing ATPase InvC at the C terminus with peptides that are recognized by bacterial tail-specific proteases. This caused a dramatic increase in InvC turnover which rendered even assembled injectisomes inactive. Bacterial strains conditionally expressing these unstable InvC variants were proficient for invasion but underwent rapid and sustained intracellular inactivation of T3SS1 activity when InvC expression ceased. This allowed us to directly implicate T3SS1 activity in cytosolic colonization and bacterial egress. We subsequently identified two T3SS1-delivered effectors, SopB and SipA, that are required for efficient colonization of the epithelial cell cytosol. Overall, our findings support a multifaceted, postinvasion role for T3SS1 and its effectors in defining the cytosolic population of intracellular SalmonellaIMPORTANCE A needle-like apparatus, the type III secretion system (T3SS) injectisome, is absolutely required for Salmonella enterica to enter epithelial cells; this requirement has hampered the analysis of its postentry contributions. To identify T3SS1-dependent intracellular activities, in this study we overcame this limitation by developing a conditional inactivation in the T3SS whereby T3SS activity is chemically induced during culture in liquid broth, permitting bacterial entry into epithelial cells, but is quickly and perpetually inactivated in the absence of inducer. In this sense, the mutant acts like wild-type bacteria when extracellular and as a T3SS mutant once it enters a host cell. This "conditional" mutant allowed us to directly link activity of this T3SS with nascent vacuole lysis, cytosolic proliferation, and cellular egress, demonstrating that the invasion-associated T3SS also contributes to essential intracellular stages of the S. enterica infectious cycle.

    View details for DOI 10.1128/mBio.01931-17

    View details for PubMedID 29208746

    View details for PubMedCentralID PMC5717391

  • Functional relatedness in the Inv/Mxi-Spa type III secretion system family. Molecular microbiology Klein, J. A., Dave, B. M., Raphenya, A. R., McArthur, A. G., Knodler, L. A. 2017; 103 (6): 973–91

    Abstract

    Type III Secretion Systems (T3SSs) are structurally conserved nanomachines that span the inner and outer bacterial membranes, and via a protruding needle complex contact host cell membranes and deliver type III effector proteins. T3SS are phylogenetically divided into several families based on structural basal body components. Here we have studied the evolutionary and functional conservation of four T3SS proteins from the Inv/Mxi-Spa family: a cytosolic chaperone, two hydrophobic translocators that form a plasma membrane-integral pore, and the hydrophilic 'tip complex' translocator that connects the T3SS needle to the translocon pore. Salmonella enterica serovar Typhimurium (S. Typhimurium), a common cause of food-borne gastroenteritis, possesses two T3SSs, one belonging to the Inv/Mxi-Spa family. We used invasion-deficient S. Typhimurium mutants as surrogates for expression of translocator orthologs identified from an extensive phylogenetic analysis, and type III effector translocation and host cell invasion as a readout for complementation efficiency, and identified several Inv/Mxi-Spa orthologs that can functionally substitute for the S. Typhimurium chaperone and translocator proteins. Functional complementation correlates with amino acid sequence identity between orthologs, but varies considerably between the four proteins. This is the first in-depth survey of the functional interchangeability of Inv/Mxi-Spa T3SS proteins acting directly at the host-pathogen interface.

    View details for DOI 10.1111/mmi.13602

    View details for PubMedID 27997726

  • Measurement of Salmonella enterica Internalization and Vacuole Lysis in Epithelial Cells. Methods in molecular biology (Clifton, N.J.) Klein, J. A., Powers, T. R., Knodler, L. A. 2017; 1519: 285–96

    Abstract

    Establishment of an intracellular niche within mammalian cells is key to the pathogenesis of the gastrointestinal bacterium, Salmonella enterica serovar Typhimurium (S. Typhimurium). Here we will describe how to study the internalization of S. Typhimurium into human epithelial cells using the gentamicin protection assay. The assay takes advantage of the relatively poor penetration of gentamicin into mammalian cells; internalized bacteria are effectively protected from its antibacterial actions. A second assay, the chloroquine (CHQ) resistance assay, can be used to determine the proportion of internalized bacteria that have lysed or damaged their Salmonella-containing vacuole and are therefore residing within the cytosol. Its application to the quantification of cytosolic S. Typhimurium in epithelial cells will also be presented. Together, these protocols provide an inexpensive, rapid and sensitive quantitative measure of bacterial internalization and vacuole lysis by S. Typhimurium.

    View details for DOI 10.1007/978-1-4939-6581-6_19

    View details for PubMedID 27815887

  • The type III secretion system apparatus determines the intracellular niche of bacterial pathogens. Proceedings of the National Academy of Sciences of the United States of America Du, J., Reeves, A. Z., Klein, J. A., Twedt, D. J., Knodler, L. A., Lesser, C. F. 2016; 113 (17): 4794–99

    Abstract

    Upon entry into host cells, intracellular bacterial pathogens establish a variety of replicative niches. Although some remodel phagosomes, others rapidly escape into the cytosol of infected cells. Little is currently known regarding how professional intracytoplasmic pathogens, including Shigella, mediate phagosomal escape. Shigella, like many other Gram-negative bacterial pathogens, uses a type III secretion system to deliver multiple proteins, referred to as effectors, into host cells. Here, using an innovative reductionist-based approach, we demonstrate that the introduction of a functional Shigella type III secretion system, but none of its effectors, into a laboratory strain of Escherichia coli is sufficient to promote the efficient vacuole lysis and escape of the modified bacteria into the cytosol of epithelial cells. This establishes for the first time, to our knowledge, a direct physiologic role for the Shigella type III secretion apparatus (T3SA) in mediating phagosomal escape. Furthermore, although protein components of the T3SA share a moderate degree of structural and functional conservation across bacterial species, we show that vacuole lysis is not a common feature of T3SA, as an effectorless strain of Yersinia remains confined to phagosomes. Additionally, by exploiting the functional interchangeability of the translocator components of the T3SA of Shigella, Salmonella, and Chromobacterium, we demonstrate that a single protein component of the T3SA translocon-Shigella IpaC, Salmonella SipC, or Chromobacterium CipC-determines the fate of intracellular pathogens within both epithelial cells and macrophages. Thus, these findings have identified a likely paradigm by which the replicative niche of many intracellular bacterial pathogens is established.

    View details for DOI 10.1073/pnas.1520699113

    View details for PubMedID 27078095

    View details for PubMedCentralID PMC4855615

  • Intracellular accumulation dynamics and fate of zinc ions in alveolar epithelial cells exposed to airborne ZnO nanoparticles at the air-liquid interface. Nanotoxicology Mihai, C., Chrisler, W. B., Xie, Y., Hu, D., Szymanski, C. J., Tolic, A., Klein, J. A., Smith, J. N., Tarasevich, B. J., Orr, G. 2015; 9 (1): 9–22

    Abstract

    Airborne nanoparticles (NPs) that enter the respiratory tract are likely to reach the alveolar region. Accumulating observations support a role for zinc oxide (ZnO) NP dissolution in toxicity, but the majority of in-vitro studies were conducted in cells exposed to NPs in growth media, where large doses of dissolved ions are shed into the exposure solution. To determine the precise intracellular accumulation dynamics and fate of zinc ions (Zn(2+)) shed by airborne NPs in the cellular environment, we exposed alveolar epithelial cells to aerosolized NPs at the air-liquid interface (ALI). Using a fluorescent indicator for Zn(2+), together with organelle-specific fluorescent proteins, we quantified Zn(2+) in single cells and organelles over time. We found that at the ALI, intracellular Zn(2+) values peaked 3 h post exposure and decayed to normal values by 12 h, while in submerged cultures, intracellular Zn(2+) values continued to increase over time. The lowest toxic NP dose at the ALI generated peak intracellular Zn(2+) values that were nearly three-folds lower than the peak values generated by the lowest toxic dose of NPs in submerged cultures, and eight-folds lower than the peak values generated by the lowest toxic dose of ZnSO4 or Zn(2+). At the ALI, the majority of intracellular Zn(2+) was found in endosomes and lysosomes as early as 1 h post exposure. In contrast, the majority of intracellular Zn(2+) following exposures to ZnSO4 was found in other larger vesicles, with less than 10% in endosomes and lysosomes. Together, our observations indicate that low but critical levels of intracellular Zn(2+) have to be reached, concentrated specifically in endosomes and lysosomes, for toxicity to occur, and point to the focal dissolution of the NPs in the cellular environment and the accumulation of the ions specifically in endosomes and lysosomes as the processes underlying the potent toxicity of airborne ZnO NPs.

    View details for DOI 10.3109/17435390.2013.859319

    View details for PubMedID 24289294

    View details for PubMedCentralID PMC4652791

  • Vaccination with a UV-irradiated genetically attenuated mutant of Staphylococcus aureus provides protection against subsequent systemic infection. The Journal of infectious diseases Burnside, K., Lembo, A., Harrell, M. I., Klein, J. A., Lopez-Guisa, J., Siegesmund, A. M., Torgerson, T. R., Oukka, M., Molina, D. M., Rajagopal, L. 2012; 206 (11): 1734–44

    Abstract

    Staphylococcus aureus are gram-positive bacteria that cause clinically significant infections in humans. Severe S. aureus infections are particularly problematic in hospitalized patients and reoccur despite therapeutic measures. The absence of natural protective immune responses and the lack of high-throughput approaches to identify S. aureus antigens have imposed constraints in the development of effective vaccines. Here, we showed that vaccination with the genetically attenuated S. aureus mutant, inactivated using UV irradiation rather than heat, significantly increased survival and diminished bacterial burden and kidney abscesses when mice were challenged with virulent methicillin-sensitive or methicillin-resistant S. aureus. Protection conferred by immunization could be transferred to the naive host and was not observed in B-cell-deficient mice. Using a novel S. aureus whole-proteome microarray, we show that immunoglobulin G antibody responses to 83 proteins were observed in the immunized mice. These results suggest that protection against S. aureus infections requires antibody responses to the wide repertoire of antigens/virulence factors. Vaccination using UV-irradiated genetically attenuated S. aureus induces humoral immunity and provides a vaccine strategy for pathogens that fail to induce protective immunity. We also describe a novel, high-throughput technology to easily identify S. aureus antigens for vaccine development.

    View details for DOI 10.1093/infdis/jis579

    View details for PubMedID 22966130

    View details for PubMedCentralID PMC3488195